Compositions and methods for monitoring construction of mammalian synthetic chromosomes in real time and for bioengineering mammalian synthetic chromosomes

Abstract

To provide synthetic chromosomes, and to provide eukaryotic host cells containing synthetic chromosomes.SOLUTION: The present invention encompasses compositions and methods to allow one to monitor formation of synthetic chromosomes in real-time via standardized fluorescent technology, eliminating the need for cumbersome, expensive, and possibly mutagenic analysis. The synthetic chromosome of the present invention comprises a site for binding at least two differently labeled tags, the first tag of the two tags detects a first site present on both a synthetic chromosome and an endogenous chromosome, and a second synthetic chromosome-specific tag identifies a second site present on the synthetic chromosome apart from the endogenous chromosomes.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The field of the invention includes compositions and methods that allow for real-time monitoring of the production of synthetic chromosomes by standardized microscopy. [Background technology]

[0002] In the following discussion, for purposes of background and introductory text, certain articles and methods will be described. Nothing contained herein should be construed as an "admission" of prior art. Applicants expressly reserve the right, where necessary, to demonstrate that the articles and methods referenced herein do not constitute prior art under applicable statutory provisions.

[0003] The ability to generate fully functional mammalian synthetic chromosomes represents a powerful system for cell-based correction of genetic disorders, production of recombinant proteins in transgenic animals, analysis of the regulation and expression of numerous human genes in various cell types and in animal models of human disease, studies on meiosis and chromosome structure, directing cellular differentiation and de-differentiation, generating induced pluripotent stem cells, creating novel autocrine and paracrine cellular signaling networks, generating multiple expression systems capable of stoichiometrically producing multiple encoded factors, producing biological circuits, inserting DNA elements capable of probing the nuclear structure and using interactions discovered within the nuclear structure downstream to regulate expression of the genome, and manipulating large DNA elements, such as, but not limited to, chromosome arm exchanges on synthetic chromosomes or the incorporation of multiple large DNA elements on synthetic chromosomes.

[0004] Fully functional mammalian synthetic chromosomes have several advantages over virus-based delivery systems, such as increased payload size, the fact that extrachromosomal maintenance avoids potential host cell destruction, avoids transcriptional silencing of introduced genes and potential immunological complications, and mammalian synthetic chromosomes can originate from and be tailored to the species into which they are to be inserted. However, while successful production of mammalian synthetic chromosomes has been demonstrated, confirming successful chromosome production requires extensive screening time and effort to identify and characterize the synthetic chromosomes of interest. The synthetic chromosome production process is ad hoc in nature. Thus, there is a need in the art for compositions and methods that allow for real-time tracking of the production of new synthetic chromosomes as they are being generated. The present invention provides methods and compositions that address this need. Summary of the Invention [Problem to be solved by the invention]

[0005] There is a need in the art for compositions and methods that allow for real-time tracking of the production of new synthetic chromosomes as they are being generated. [Means for solving the problem]

[0006] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will become apparent from the Detailed Description set forth below, including the aspects illustrated in the accompanying drawings and defined in the appended claims.

[0007] In some embodiments, the present invention provides a method for screening the production of synthetic chromosomes, comprising the steps of constructing a synthetic chromosome production reporter cell line comprising an endogenous chromosome tag and a synthetic chromosome tag, wherein the endogenous chromosome tag and the synthetic chromosome tag become stably integrated into the synthetic chromosome production reporter cell line genome; transfecting the synthetic chromosome production reporter cell line with synthetic chromosome production components; and monitoring the production of synthetic chromosomes in the synthetic chromosome production reporter cell line.

[0008] In yet another embodiment, the present invention provides a method for screening the production of synthetic chromosomes, comprising the steps of: providing a synthetic chromosome production reporter cell line; transfecting the synthetic chromosome production reporter cell line with synthetic chromosome production components and endogenous chromosome tags and synthetic chromosome tags, wherein the endogenous chromosome tags and synthetic chromosome tags become stably integrated into the synthetic chromosomes; and monitoring the production of synthetic chromosomes in the synthetic chromosome production reporter cell line.

[0009] In yet another embodiment, the present invention provides a method for screening the production of synthetic chromosomes, comprising the steps of constructing a synthetic chromosome production reporter cell line comprising an endogenous chromosome tag, wherein the endogenous chromosome tag becomes stably integrated into the genome of the synthetic chromosome production reporter cell line; transfecting the synthetic chromosome production reporter cell line with synthetic chromosome production components and the synthetic chromosome tag, wherein the synthetic chromosome tag becomes stably integrated into the synthetic chromosome; and monitoring the production of synthetic chromosomes in the synthetic chromosome production reporter cell line.

[0010] In yet another embodiment, the present invention provides a method for screening the production of synthetic chromosomes, comprising the steps of constructing a synthetic chromosome production reporter cell line comprising a synthetic chromosome tag, wherein the synthetic chromosome tag becomes stably integrated into the genome of the synthetic chromosome production reporter cell line; transfecting the synthetic chromosome production reporter cell line with synthetic chromosome production components and an endogenous chromosome tag, wherein the endogenous chromosome tag becomes stably integrated into the synthetic chromosome; and monitoring the production of synthetic chromosomes in the synthetic chromosome production reporter cell line.

[0011] In some aspects of the foregoing embodiments, arms of endogenous chromosomes present in the reporter cell line comprise recombination sites that are compatible for interaction with recombination sites in the synthetic chromosome. Furthermore, in some aspects of the foregoing embodiments, the tags of the endogenous chromosomal tags and the synthetic chromosomal tags are fluorescent tags, and the monitoring step is performed by fluorescence microscopy. In some configurations of this embodiment, the fluorescent tag is selected from TagBFP, TagCFP, TagGFP2, TagYFP, TagRFP, FusionRed, mKate2, TurboGFP, TurboYFP, TurboRFP, TurboFP602, TurboFP635, TurboFP650, AmCyan1, AcvGFP1, ZsGreen1, ZsYellow1, mBanana, mOrange, mOrange2, DsRed-Express2, DsRed-Express, tdTomato, DsRed-Monomer, DsRed2, AsRed2, mStrawberry, mCherry, HcRed1, mRaspberry, E2-Crimson, mPlum, Dendra2, Timer, and PAmCherry, HALO-tag, or infrared-shifted fluorescent proteins.

[0012] In other aspects of the above embodiments, the tags of the endogenous chromosomal tags and the synthetic chromosomal tags are chemiluminescent tags and the monitoring step is performed by chemiluminescent microscopy, or are phosphorescent tags and the monitoring step is performed by phosphorescence microscopy.

[0013] In some aspects of the foregoing embodiments, the endogenous chromosomal tags comprise markers specific for histones H1, H2A, H2B, H3, H4, or H5, and the synthetic chromosomal tags comprise markers specific for histones H1, H2A, H2B, H3, H4, or H5 that have been screened against a database of known sequences. n It comprises nucleotides.

[0014] In some aspects of the foregoing embodiments, the synthetic chromosome-producing reporter cell line is selected from a mammalian cell line, an embryonic cell line, a pluripotent cell line, an adult-derived stem cell, a reprogrammed cell line or a human cell line, and in a preferred aspect, the synthetic chromosome-producing reporter cell line is the human cell line HT1080.

[0015] In some aspects of embodiments of the invention, synthetic chromosome production reporter cell lines are transfected with synthetic chromosome production components to produce synthetic chromosomes in a top-down manner, in other aspects, synthetic chromosome production reporter cell lines are transfected with synthetic chromosome production components to produce synthetic chromosomes in a bottom-up manner, in still other aspects, synthetic chromosome production reporter cell lines are transfected with synthetic chromosome production components to produce synthetic chromosomes by genetic engineering of naturally occurring minichromosomes, and in preferred aspects, synthetic chromosome production reporter cell lines are transfected with synthetic chromosome production components to produce synthetic chromosomes by de novo chromosome generation by targeted amplification of a chromosome segment, and in a preferred method of this aspect, the chromosome segment is a pericentric region of a chromosome.

[0016] In some aspects of the foregoing embodiments, the endogenous chromosomal tag comprises a plurality of endogenous chromosomal tags. In some aspects of the foregoing embodiments, the endogenous chromosomal tag is a fusion protein, a nucleic acid / protein chimera, a nucleic acid / protein complex (e.g., RNA / CRISPR-CAS9), or a moiety comprising a TALEN protein specific for an endogenous chromosome and a fluorescent or phosphorescent label, and the synthetic chromosomal tag is a nucleic acid / protein chimera.

[0017] The present invention further provides a synthetic chromosome-producing reporter cell line comprising an endogenous chromosomal tag and a synthetic chromosomal tag, wherein the endogenous chromosomal tag and the synthetic chromosomal tag are stably integrated into the genome of the synthetic chromosome-producing reporter cell line.

[0018] Additionally, the present invention further provides synthetic chromosomes produced by synthetic chromosome producing cell lines, and synthetic chromosomes whose production has been monitored in real time by the methods of the present invention.

[0019] These and other aspects and uses of the present invention will be explained in the detailed description. Effect of the Invention

[0020] In accordance with the present invention, there are provided synthetic chromosomes and eukaryotic host cells containing the synthetic chromosomes. [Brief description of the drawings]

[0021] [Figure 1] 1 is a simplified flow chart showing method steps for creating a system for tracking the production of new synthetic chromosomes in real time. In this embodiment, the components of the tracking system are incorporated into the endogenous genome of a production cell line. [Diagram 2]1 is a simplified flow chart showing method steps for creating an alternative system for tracking the production of new synthetic chromosomes in real time, in which components of the tracking system are incorporated into the synthetic chromosomes and thus co-delivered with the synthetic chromosomes into the production cell line. [Diagram 3] 1 is a simplified flow chart showing method steps for creating yet another example system for tracking the production of new synthetic chromosomes in real time, in which one component of the tracking system is incorporated into the synthetic chromosome and one component of the tracking system is incorporated into the endogenous genome of a production cell line. [Figure 4] FIG. 1 is a simplified flow chart showing method steps for monitoring the exchange of "chromosome arms" from an endogenous genome into a synthetic chromosome. [Diagram 5] FIG. 1 shows details of one exemplary process for producing synthetic chromosomes in a synthetic chromosome-producing cell line (in this case via artificial de novo chromosome generation by targeted amplification of specific chromosome segments), which is monitored by the methods of the present invention. [Figure 6] FIG. 1 shows simplified constructs of endogenous chromosome-specific tag vectors, synthetic chromosome-specific tag vectors, and mixed endogenous and synthetic chromosome-specific vectors. [Figure 7] FIG. 1 shows a timeline of real-time synthetic chromosome production using the methods of the present invention. [Figure 8] Schematic representation of the use of the CRISPR / Cas fluorescent visualization system to monitor, isolate, and / or purify synthetic chromosomes. [Figure 9] Schematic representation of the use of two different CRISPR / Cas fluorescent visualization systems to monitor, isolate, and / or purify synthetic chromosomes. [Figure 10] Schematic representation of the use of two different CRISPR / Cas fluorescent visualization systems to monitor chromosome arm exchange between endogenous and synthetic chromosomes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The methods described herein may employ conventional techniques and explanations of molecular biology (including recombinant techniques), cell biology, biochemistry, and cell engineering techniques, all of which are within the skill of the art, unless otherwise indicated. Such conventional techniques include oligonucleotide synthesis, oligonucleotide hybridization and ligation, cell transformation and transduction, recombinant system engineering, the production of transgenic animals and plants, and human gene therapy. Specific illustrations of suitable techniques may be obtained by reference to the examples herein. However, equivalent conventional procedures may of course also be used. Such conventional techniques and explanations can be found in standard laboratory manuals, such as Green et al., eds., Genome Analysis: A Laboratory Manual Series (Vols. I-IV), 1999; Weiner et al., eds., Genetic Variation: A Laboratory Manual, 2007; Sambrook and Russell, Condensed Protocols from Molecular Cloning: A Laboratory Manual, 2006; and Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 2002 (all from Cold Spring Harbor Laboratory Press); Bollag et al., Protein Methods, John Wiley & Sons, 2003. and Sons, 1996; Wagner et al., eds., Nonviral Vectors for Gene Therapy, Academic Press, 1999;Kaplift and Loewy, eds., Viral Vectors, Academic Press, 1995; Lefkovits, ed., Immunology Methods Manual, Academic Press, 1997; Meager, ed., Gene Therapy Techniques, Applications and Regulations From Laboratory to Clinic, John Wiley & Sons, 1999; M. Giacca, Gene Therapy, Springer, 2010; LeDoux, ed., Gene Therapy Protocols, Doyle and Griffiths, eds., Cell and Tissue Culture: Laboratory Procedures in Biotechnology, John Wiley & Sons, 1998; G. Hadlaczky, ed., Mammalian Chromosome Engineering-Methods and Protocols, Humana Press, 2011; Lanza and Klimanskaya, eds., Essential Stem Cell Methods, Academic Press, 2011;Board on Health Sciences Policy, Stem Cell Therapies: Opportunities for Ensuring the Quality and Safety of Clinical Offerings: Summary of a Joint Workshop, National Academies Press, 2014; Lanza and Atala, eds., Essentials of Stem Cell Biology, 3rd ed., Academic Press, 2013; and Atala and Lanza, eds., Handbook of Stem Cells, Academic Press, 2012, all of which are incorporated by reference in their entireties for all purposes. Before the compositions, research tools and methods of the present invention are described, it is to be understood that the specific methods, compositions, targets and applications described may, of course, vary and the present invention is not limited thereto. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

[0023] As used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" refers to a composition or mixture of compositions, reference to "an assay" includes reference to equivalent steps and methods known to those skilled in the art, and so forth.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated by reference into this application for the purpose of describing and disclosing the devices, formulations and methodologies described in the publications and that may be used in connection with the presently described invention.

[0025] When a range of values ​​is provided, it will be understood that each value between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller range, subject to any specifically excluded limit in the stated range. When a stated range includes both limits, a range excluding only one of those included limits is also included in the invention.

[0026] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of ordinary skill in the art upon reading this specification that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art are not described in order to avoid obscuring the present invention.

[0027] [Definition] Unless expressly specified, terms used herein are intended to have their plain and ordinary meanings as understood by those of ordinary skill in the art. The following definitions are intended to aid the reader in understanding the invention, but are not intended to alter or otherwise limit the meaning of such terms unless specifically stated.

[0028] "Binding" as used herein (e.g., with respect to a nucleic acid binding domain of a polypeptide) refers to a non-covalent interaction between a polypeptide and a nucleic acid. During the non-covalent interaction, the polypeptide and the nucleic acid are said to be "associated," "interacting," or "bound." Binding interactions generally occur over a 10 -6 M~10 -15 It is characterized by a dissociation constant (Kd) less than M. "Affinity" refers to the strength of binding, with high binding affinity being correlated with a lower Kd.

[0029] By "binding domain" is meant a domain of a polypeptide or protein that is capable of non-covalently binding to another molecule, e.g., a binding domain can bind to a DNA molecule (a DNA-binding protein), an RNA molecule (an RNA-binding protein), and / or a protein molecule (a protein-binding protein).

[0030] A "centromere" is any nucleic acid sequence that confers the ability of a chromosome to separate into daughter cells upon cell division. A centromere can provide for stable segregation of a synthetic chromosome containing a nucleic acid sequence, e.g., a centromere, through mitosis and meiosis. A centromere does not necessarily have to be from the same species as the cell into which it is introduced, but it is preferred that the centromere has the ability to facilitate DNA segregation in cells of that species. A "dicentric" chromosome is one that contains two centromeres. A "formerly dicentric chromosome" is a chromosome that results when a dicentric chromosome fragments. A "chromosome" is a nucleic acid molecule (and associated proteins) that can replicate and separate within a cell upon cell division. Typically, a chromosome contains a centromeric region, an origin of replication, a telomeric region, and a region of nucleic acid between the centromeric and telomeric regions. An "acrocentric chromosome" refers to a chromosome with arms of unequal length.

[0031] A "coding sequence" or a sequence that "encodes" a peptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) in vivo to produce a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are typically determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.

[0032] The term DNA "control sequences" refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites, enhancers, and the like, which collectively provide for the replication, transcription, and translation of a coding sequence in a recipient cell. Not all of the above types of control sequences need be present so long as the selected coding sequence is capable of being replicated, transcribed, and translated in an appropriate host cell.

[0033] "Endogenous chromosome" refers to the chromosome found in a cell prior to the creation or introduction of a synthetic chromosome. As used herein, "euchromatin" refers to chromatin that is diffusely stained and typically contains genes, and "heterochromatin" refers to chromatin that remains abnormally condensed and is considered transcriptionally inactive. Highly repetitive sequence DNA (satellite DNA) is usually located in regions of heterochromatin surrounding the centromere.

[0034] The terms "heterologous DNA" or "foreign DNA" (or "heterologous RNA" or "foreign RNA") are used interchangeably and refer to DNA or RNA that is not naturally occurring as part of the genome in which it resides, or that is found in a genome or cell in a different location and / or amount than the DNA or RNA occurs in nature. Examples of heterologous DNA include, but are not limited to, DNA that encodes a gene product or gene products of interest. Other examples of heterologous DNA include, but are not limited to, DNA that encodes traceable marker proteins and regulatory DNA sequences.

[0035] "Operably linked" refers to an arrangement of elements in which the components so expressed are configured to perform their normal function. Thus, a control sequence operably linked to a coding sequence can effect expression of the coding sequence. A control sequence need not be contiguous with the coding sequence, so long as the sequence functions to direct expression of the coding sequence. Thus, for example, a non-translated but transcribed intervening sequence can be present between a promoter sequence and a coding sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence. Indeed, such sequences need not be present on the same contiguous DNA molecule (i.e., chromosome), and can still interact to effect regulatory changes.

[0036] A "promoter" or "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a polynucleotide or polypeptide coding sequence, such as messenger RNA, ribosomal RNA, small nuclear of nucleolar RNA, or any type of RNA transcribed by any of the classes of RNA polymerase I, II, or III.

[0037] A "recognition sequence" is a specific sequence of nucleotides that is recognized and bound by a protein, DNA or RNA molecule, or combination thereof (such as, but not limited to, a restriction endonuclease, a modification methylase, or a recombinase). For example, the recognition sequence for Cre recombinase is a 34 base pair sequence that contains two 13 base pair inverted repeats (which serve as recombinase binding sites) flanking an 8 base pair core, called loxP (see, e.g., Sauer, Current Opinion in Biotechnology, 1994, vol. 5, pp. 521-527). Other examples of recognition sequences include, but are not limited to, attB and attP, attR and attL, which are recognized by the recombinase enzyme bacteriophage lambda integrase, as well as others. The recombination site, called attB, is an approximately 33 base pair sequence that contains two 9 base pair core-type Int binding sites and a 7 base pair overlap region; attP is an approximately 240 base pair sequence that contains a core-type Int binding site and an arm-type Int binding site as well as sites for the auxiliary proteins IHF, FIS and Xis (see, e.g., Landy, Current Opinion in Biotechnology, 1993, 3:699-7071).

[0038] "Recombinase" refers to an enzyme that catalyzes the exchange of DNA segments at specific recombination sites. Integrases refer to recombinases that are usually derived from viruses or transposons, and possibly ancient viruses. "Recombination proteins" include excision proteins, integrative proteins, enzymes, cofactors, and associated proteins that participate in recombination reactions with one or more recombination sites (see Landy, Current Opinion in Biotechnology, 1993, vol. 3, pp. 699-707). The recombination proteins used in the methods herein can be delivered to cells via expression cassettes on suitable vectors, such as plasmids. In other embodiments, the recombination proteins can be delivered to cells in the form of proteins in the same reaction mixture used to deliver the desired nucleic acid. In yet other embodiments, the recombinases can be encoded within the cells and expressed as needed using highly regulated inducible promoters.

[0039] "Ribosomal RNA" (rRNA) is a specialized RNA that forms part of the structure of the ribosome and is involved in the synthesis of proteins. Ribosomal RNA is produced by transcription of genes that exist in multiple copies in eukaryotic cells. In human cells, approximately 250 copies of rRNA genes (i.e. genes that code for rRNA) per haploid genome are scattered in clusters on at least five different chromosomes (chromosomes 13, 14, 15, 21 and 22). In human cells, multiple copies of highly conserved rRNA genes are located in a series of tandemly arranged rDNA units that are generally about 40-45 kb in length and contain transcribed and non-transcribed regions known as spacer (i.e. intergenic spacer) DNA that vary in length and sequence.

[0040] As used herein, the term "selection marker" refers to a gene that is introduced into a cell, particularly in the context of the present invention, into a cell in culture, that confers a trait suitable for artificial selection. Commonly used selection markers are well known to those skilled in the art. In a preferred embodiment, the selection markers used in the human synthetic chromosome system should be non-immunogenic in humans and include, but are not limited to: human nerve growth factor receptor (detected with MAbs as described in U.S. Pat. No. 6,365,373); truncated human growth factor receptor (detected with MAbs); mutant human dihydrofolate reductase (DHFR; available with a fluorescent MTX substrate); secreted alkaline phosphatase (SEAP; available with a fluorescent substrate); human thymidylate synthase (TS; confers resistance to the anti-cancer drug fluorodeoxyuridine); human glutathione S-transferase alpha (GSTA1; conjugates glutathione to busulfan, a stem cell-selective alkylating agent; CD34 + Selection markers that have a chemoprotective effect on cells; CD24 cell surface antigen in hematopoietic stem cells; human CAD gene that confers resistance to N-phosphonoacetyl-L-aspartic acid (PALA); human multidrug resistance 1 (MDR-1; P-glycoprotein surface protein that can be selected for increased drug resistance or enriched by FACS); human CD25 (IL-2α; detectable by Mab-FITC); methylguanine DNA methyltransferase (MGMT; selectable by carmustine); and cytidine deaminase (CD; selectable by Ara-C). Drug selection markers such as puromycin, hygromycin, blasticidin, G418, tetracycline can also be used. In addition, using FACs sorting, any fluorescent marker gene may be used for positive selection, such as when a chemiluminescent marker (e.g. Halotag®) is used. Cell surface proteins linked to moieties that can be bound to magnetic beads or surfaces to isolate or enrich the cells of interest are additional selection marker mechanisms.

[0041] "Site-specific recombination" refers to site-specific recombination that takes place between two specific sites on a single nucleic acid molecule or between two different molecules, and requires the presence of an exogenous protein, such as an integrase or recombinase. Certain site-specific recombination systems can be used to perform specific deletions, inversions, or insertions of DNA, with precise events controlled by the orientation of specific sites, specific systems, and the presence of auxiliary proteins or factors. In addition, segments of DNA can be exchanged between chromosomes (chromosome arm exchange), as described in Figure 4.

[0042] A "synthetic chromosome" (also called an "artificial chromosome") is a nucleic acid molecule, typically DNA, that stably replicates and segregates together with endogenous chromosomes in cells that have the ability to accept and express heterologous genes. A "mammalian synthetic chromosome" refers to a chromosome that has an active mammalian centromere. A "human synthetic chromosome" refers to a chromosome with a centromere that functions in, and is preferably produced in, human cells. For exemplary artificial chromosomes, see, e.g., U.S. Patent Nos. 8,389,802; 7,521,240; 6,025,155; 6,077,697; 5,891,691; 5,869,294; 5,721,118; 5,712,134; 5,695,967; and 5,288,625, and published PCT applications WO 97 / 40183 and WO 98 / 08964.

[0043] The terms "subject," "individual," or "patient" can be used interchangeably herein and can refer to a mammal, and in some embodiments, a human. A "vector" is a replicon, such as a plasmid, phage, viral construct, cosmid, bacterial artificial chromosome, P-1 derived artificial chromosome, or yeast artificial chromosome, into which another DNA segment can be attached. In some instances, the vector may be a chromosome, such as in the case of arm swapping from one endogenous chromosome to a synthetic chromosome engineered to contain recombination sites. Vectors are used to transduce and express DNA segments into cells.

[0044] [The present invention] The present invention encompasses compositions and methods that allow for the formation of synthetic chromosomes to be monitored in real time by standardized microscopic methods such as fluorescence microscopy or other visual methods, eliminating the need for cumbersome, expensive, and possibly mutagenic analyses. Prior to the present invention, the production of synthetic chromosomes was essentially ad hoc, with the production of new synthetic chromosomes unable to be tracked in real time and success only being measured after colonies of drug-resistant cells were cloned, grown, and analyzed by low-throughput methods such as fluorescent in situ hybridization (FISH) screening of large numbers of clones.

[0045] Advantages of the compositions and methods of the invention include: 1) synthetic chromosome production can be monitored in real time by standardized fluorescent microscopy (or other visual means), allowing for rapid elimination of unsuccessful clones (i.e., drug-resistant colonies that did not produce synthetic chromosomes); 2) the need for periodic FISH analysis to test for chromosome formation is reduced or eliminated; 3) the system can be used to test components for the generation of synthetic chromosomes and the stoichiometry of the test components can be assessed, leading to optimization of synthetic chromosome production; 4) the system provides an assayable format that allows for screening of potential small molecule compounds that control or enhance synthetic chromosome production; 5) the need for potentially mutagenic dyes to identify synthetic chromosomes during flow cytometry-based sorting, isolation and transfer is reduced or eliminated; and 6) the reduction or elimination of screening steps reduces the cost of reagents required and the time required to characterize synthetic chromosomes. For example, Lindenbaum and Perkins et al., Nucleic Acid Research, 2004, vol. 32, no. 21, e172, describe the production of an artificial chromosome expression (ACE) system based on mammalian satellite DNA. This system did not allow real-time tracking of new synthetic chromosomes, and success could only be measured after colonies of drug-resistant cells were cloned and expanded over a period of approximately three months, and a large number of clones were analyzed by fluorescent in situ hybridization (FISH) screening. The present invention makes it possible to reduce or completely eliminate repeated FISH analysis, which requires at least four days to perform.

[0046] The compositions and methods of the invention for real-time monitoring of synthetic chromosome production are applicable to all methodologies of synthetic chromosome production, including "top-down" approaches, "bottom-up" approaches, genetic engineering of naturally occurring minichromosomes, and artificial de novo chromosome generation by targeted amplification of specific chromosome segments (all of which are discussed in more detail below).

[0047] Figure 1 is a simplified flow chart showing the method steps of one embodiment for creating a system for tracking the production of new synthetic chromosomes in real time. The first step 101 of the method 100 is to construct a synthetic chromosome-producing reporter cell line in which the synthetic chromosomes will be produced. The construction of a synthetic chromosome-producing reporter cell line essentially requires stably transforming a cell line to express at least two defined tags: one labeled tag specific to the endogenous chromosome of the chromosome-engineered reporter cell line, and one differently labeled tag specific to a sequence on the synthetic chromosome to be produced (i.e., a "cis-endogenous chromosome" system in which the tags are encoded by the endogenous chromosomes of the reporter cell line). The method and tags used to construct the synthetic chromosome-producing reporter cell line will vary depending on the species from which the cells are derived and the tag complement on the synthetic chromosomes to be produced.

[0048] The second step 103 is to transfect or transform the synthetic chromosome-producing reporter cell line constructed in step 101 with appropriate components for the production of synthetic chromosomes. The appropriate components will vary depending on the method of chromosome production used (i.e., whether the method is "top-down", "bottom-up", genetic engineering of minichromosomes, or de novo chromosome generation) and the system selected for engineering the genetic payload into the synthetic chromosomes.

[0049] Finally, the third step 105 is to monitor the production of synthetic chromosomes in real time, for example by fluorescence microscopy or other visual means to analyze and track the endogenous and synthetic chromosome-specific tags. The endogenous chromosome-specific tags allow the endogenous chromosome population to be assessed to ensure the "health" of the synthetic chromosome-producing reporter cell line, and the synthetic chromosome-specific tags allow the formation and continued presence of synthetic chromosomes to be assessed. In addition, if the synthetic chromosome-specific tags are specific for site-specific import sequences (acceptor sites) on the synthetic chromosomes, the signal from the labeled tags is indirectly a measure of the number of acceptor sites.

[0050] Figure 2 shows an alternative embodiment of the compositions and methods of the present invention. Similar to Figure 1, Figure 2 is a simplified flow chart showing the method steps of one embodiment for creating a system for tracking the production of new synthetic chromosomes in real time. The first step 201 of the method 200 is to select a synthetic chromosome producing cell line in which to produce the synthetic chromosomes. It is noted that whereas in the previous embodiment the reporter cell line was constructed to express two defined reporter tags ("endogenous chromosomes in cis"), in this alternative embodiment the synthetic chromosome producing cell line does not express the reporter tags; instead the synthetic chromosomes are engineered to express the reporter tags (i.e., "synthetic chromosomes in cis" where the tags are encoded by the synthetic chromosomes).

[0051] The second step 203 is to transfect the synthetic chromosome producing cell line constructed in step 201 with appropriate components for the production of synthetic chromosomes. As discussed with respect to the first embodiment, the appropriate components will vary depending on the method of chromosome production used (i.e., whether the method is "top-down", "bottom-up", genetic engineering of minichromosomes, or de novo chromosome generation) and the system selected for engineering the genetic payload into the synthetic chromosomes. However, in contrast to the first embodiment, in this alternative embodiment, the synthetic chromosomes are engineered to express two reporter tags: one labeled tag specific for an endogenous chromosome of the synthetic chromosome producing cell line, and one differently labeled tag specific for a sequence on the synthetic chromosome to be produced.

[0052] Finally, the third step 205 of method 200 is the same as the third step 105 of method 100: monitoring the production of synthetic chromosomes in real time by fluorescence microscopy or other visual means by tracking the endogenous chromosome-specific tags and the synthetic chromosome-specific tags. The endogenous chromosome-specific tags allow for the assessment of the endogenous chromosome population to ensure the "health" of the synthetic chromosome-producing cell line, and the synthetic chromosome-specific tags allow for the assessment of the formation and continued presence of the synthetic chromosomes.

[0053] This alternative method differs in that the method illustrated in Figure 1 requires the construction of a synthetic chromosome-producing reporter cell line, whereas the method of Figure 2 requires only the selection of a cell line for synthetic chromosome production without genetically engineering the cell line to have a reporting characteristic. In either method, the choice of cells that are genetically engineered to report and produce synthetic chromosomes (method of Figure 1) or cells that are used only to produce synthetic chromosomes (method of Figure 2) depends, at least in part, on the species for which the synthetic chromosomes are being produced and the type of cell to which the synthetic chromosomes will ultimately be delivered. For example, to produce synthetic chromosomes for use in humans, it is desirable to choose a human cell line for the production of the synthetic chromosomes, as a fully humanized synthetic chromosome is likely to avoid issues such as potential immune responses and genetic stability issues that synthetic chromosomes produced in non-human mammalian cells may have when delivered to a human patient.

[0054] FIG. 3 illustrates yet another alternative embodiment of the compositions and methods of the present invention. Similar to FIGS. 1 and 2, FIG. 3 is a simplified flow chart of the method steps of one embodiment for creating a system for tracking the production of new synthetic chromosomes in real time. The first step 301 of method 300 is to construct a synthetic chromosome production reporter cell line in which synthetic chromosomes are produced. The construction of a synthetic chromosome production reporter cell line essentially requires the stable transformation of a cell line to express one of two tags, a tagged tag specific to an endogenous chromosome. In step 303, the synthetic chromosome reporter cell line is transfected with the components of synthetic chromosome production and the synthetic chromosome tag to be incorporated into the synthetic chromosome. In this embodiment, the reporter cell line is engineered to express a tagged tag specific to an endogenous chromosome, and the synthetic chromosome is engineered to express a different tagged tag specific to the synthetic chromosome (i.e., a "trans-chromosome" system in which tags are expressed by different chromosomes, i.e., the endogenous chromosome and the synthetic chromosome). It should be noted that in the foregoing embodiments, either a reporter cell line was constructed to express two defined reporter tags (the "endogenous chromosome in cis" system) or a synthetic chromosome was engineered to express two defined reporter tags (the "synthetic chromosome in cis" system).

[0055] Finally, the third step 305 of method 300 is the same as the third step 105 of method 100 and 205 of method 200: monitoring the production of synthetic chromosomes in real time by fluorescence microscopy or other visual means by tracking endogenous chromosome-specific tags and synthetic chromosome-specific tags. The endogenous chromosome-specific tags allow for the assessment of the endogenous chromosome population to ensure the "health" of the synthetic chromosome-producing cell line, and the synthetic chromosome-specific tags allow for the assessment of the formation and continued presence of synthetic chromosomes.

[0056] It should be noted that it is also possible to construct a "reverse trans-chromosome" system, which is another alternative system in which a synthetic chromosome-producing reporter cell line is stably transformed to express a differently labeled tag specific to the synthetic chromosome, and the synthetic chromosome is engineered to express a labeled tag specific to the endogenous chromosome. Essentially, whether to choose the system of Figure 1 ("endogenous chromosome in cis" system), Figure 2 ("synthetic chromosome in cis system"), Figure 3 ("chromosome in trans" system) or the "reverse trans-chromosome system" is simply a design choice based on the design of the vector, the method for making the synthetic chromosome, the cell line being engineered, etc.

[0057] Once produced, the synthetic chromosomes may be used in any number of pharmacological, therapeutic and biological applications, such as, for example, producing recombinant proteins in transgenic animals, analyzing the regulation and expression of numerous human genes in various cell types or animal models, and analyzing chromosome-based gene delivery vectors for ex vivo gene therapy, directing cell differentiation and de-differentiation, forming induced pluripotent stem cells, creating novel autocrine and paracrine cell signaling networks, creating multiple expression systems capable of stoichiometrically producing multiple encoded factors, producing biological circuits, inserting DNA elements capable of probing the nuclear structure and using interactions discovered within the nuclear structure downstream to regulate expression of the genome, and engineering large DNA elements (>50Kb, >100Kb, >200Kb or more), such as, but not limited to, chromosome arm exchange onto the synthetic chromosome or the incorporation of multiple large DNA elements onto the synthetic chromosome.

[0058] 4 is a simplified flow chart of method steps showing how the method of the present invention can be used to monitor the exchange of a "chromosome arm" from an endogenous genome to a synthetic chromosome. In step 401, a synthetic chromosome production reporter cell line is constructed comprising an endogenous chromosome tag and a synthetic chromosome tag. In step 403, an arm of an endogenous chromosome of the synthetic chromosome production reporter cell line is selected for genetic engineering with a recombination site. In step 405, the synthetic chromosome production reporter cell line is transfected with components of the synthetic chromosome production, and in step 407, the exchange of the arm of the endogenous chromosome with a portion of the synthetic chromosome is monitored by determining whether a label that colors the endogenous chromosome is detected as associated with the synthetic chromosome.

[0059] <Cells that produce synthetic chromosomes> In some embodiments, the cells to be engineered and / or to produce the synthetic chromosomes may be cells naturally occurring in the subject (human patient, animal or plant) in which the genes or regulatory sequences from the synthetic chromosomes will ultimately be expressed. Such cells may be primary cell lines established for the purpose of producing synthetic chromosomes specific to an individual. In other embodiments, the cells to be engineered and / or to produce the synthetic chromosomes are derived from established cell lines. A wide variety of cell lines for tissue culture are known in the art.Examples of cell lines include, but are not limited to, human cell lines such as 293-T (embryonic kidney), 721 (melanoma), A2780 (ovarian), A172 (glioblastoma), A253 (carcinoma), A431 (epithelial), A549 (carcinoma), BCP-1 (lymphoma), BEAS-2B (lung), BR293 (breast), BxPC3 (pancreatic cancinoma), Cal-27 (tongue), COR-L23 (lung), COV-434 (ovarian), CMLT1 (leukemia), D UI45 (prostate), DuCaP (prostate), FM3 (lymph node), H1299 (lung), H69 (lung), HCA2 (fibroblast), HEK0293 (embryonic kidney), HeLa (cervical), HL-60 (myeloblast), HMEC (epithelium), HT-29 (colon), HUVEC (umbilical vein epithelium), Jurkat (T cell leukemia), JY (lymphoblastoid), K562 (lymphoblastoid), KBM-7 (lymphoblastoid), Ku812 (lymphoblastoid), KCL22 ( lymphoblastoid), KGI (lymphoblastoid), KYO1 (lymphoblastoid), LNCap (prostate), Ma-Mel (melanoma), MCF-7 (breast), MDF-10A (breast), MDA-MB-231, -468 and -435 (breast), MG63 (osteosarcoma), MOR / 0.2R (lung), MONO-MAC6 (leukocyte), MRC5 (lung), MSU1.1 (fibroblast), NCI-H69 (lung), NALM-1 (peripheral blood), NW-145 (melanoma), These include OPCN / OPCT (prostate), Peer (leukemia), Raji (B lymphoma), Saos-2 (osteosarcoma), Sf21 (ovarian), Sf9 (ovarian), SiHa (cervical cancer), SKBR3 (breast cancer), SKOV-2 (ovarian carcinoma), T-47D (breast), T84 (lung), U373 (glioblastoma), U87 (glioblastoma), U937 (lymphoma), VCaP (prostate), WM39 (skin), WT-49 (lymphoblastoid), and YAR (B cell).Rodent cell lines of interest include, but are not limited to, 3T3 (mouse fibroblasts), 4T1 (mouse mammary), 9L (rat glioblastoma), A20 (mouse lymphoma), ALC (mouse bone marrow), B16 (mouse melanoma), B35 (rat neuroblastoma), bEnd.3 (mouse brain), C2C12 (mouse myoblasts), C6 (rat glioma), CGR8 (mouse embryo), CT26 (mouse carcinoma), E14Tg2 a (mouse embryo), EL4 mouse leukemia), EMT6 / AR1 (mouse mammary), Hepa1c1c7 (mouse liver carcinoma), J558L (mouse myeloma), MC-38 (mouse adenocarcinoma), MTD-1A (mouse epithelium), RBL (rat leukemia), RenCa (mouse carcinoma), X63 (mouse lymphoma), YAC-1 (mouse Be cells), BHK-1 (hamster kidney), and CHO (hamster ovary). Useful plant cell lines include, but are not limited to, BY-2, Xan-1, GV7, GF11, GT16, TBY-AtRER1B, 3n-3, and G89 (tobacco); VR, VW, and YU-1 (grape); PAR, PAP, and PAW (pokeweed); Spi-WT, Spi-1-1, and Spi12F (spinach); PSB, PSW, and PSG (sesame); A. per, A. pas, A. plo (asparagus), and A. tetracycline. Gas); Pn and Pb (bamboo); and DG330 (soybean); embryonic cell lines; pluripotent cell lines; adult derived stem cells; reprogrammed cell lines; general animal cell lines of any species or generally embryonic or reprogrammed cells; zebrafish cell lines; primary dog ​​cells; primary horse cells; chicken DT40 cells; dog cell lines; feline cell lines; patient cell lines; and, in some preferred embodiments, the HT1080 human cell line is utilized. Potentially useful cells include any living cell, although those of eukaryotic origin are specifically contemplated. These cell lines and others are available from a variety of sources known to those skilled in the art (see, for example, the American Type Culture Collection (ATCC), Manassas, VA, USA). Cells transfected with one or more of the vectors described herein are used to establish new cell lines comprising sequences from one or more of the vectors.

[0060] <<Vector for delivering labeled tags>> The choice of vector to be used for delivery of endogenous chromosome-specific labeled tags and synthetic chromosome-specific labeled tags will depend on various factors, such as the type of cells in which propagation is desired. Certain vectors are useful for amplifying and producing large amounts of the desired DNA sequence, while other vectors are suitable for expression in cells in culture. Still other vectors are suitable for introduction and expression in cells throughout the body of an animal. The selection of an appropriate vector is well within the skill of the artisan, and many vectors are commercially available. To prepare the construct, a polynucleotide is inserted into the vector, typically by ligation of the sequence into a cleaved restriction enzyme cleavage site of the vector. Alternatively, the desired nucleotide sequence can be inserted by homologous recombination or site-specific recombination. Typically, homologous recombination is performed by attaching regions of homology to the vector on both sides of the desired nucleotide sequence (e.g., cre-lox, att sites, etc.). Nucleic acids containing such sequences can be added, for example, by ligation of oligonucleotides or by polymerase chain reaction using primers that comprise both the homologous region and a portion of the desired nucleotide sequence. Exemplary vectors that may be used include, but are not limited to, those derived from recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA. For example, plasmid vectors such as pBR322, pUC19 / 18, pUC118, 119, and M13mp series vectors may be used. Bacteriophage vectors may include λgt10, λgt11, λgt18-23, λZAP / R, and EMBL series bacteriophage vectors. Cosmid vectors that may be utilized include, but are not limited to, pJB8, pCV103, pCV107, pCV108, pTM, pMCS, pNNL, pHSG274, COS202, COS203, pWE15, pWE16, and Charomid 9 series vectors.Further vectors include bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and P1-derived artificial chromosomes (PACS), which are DNA constructs derived from the DNA of the P1 bacteriophage, based on a functional fertility plasmid (F plasmid).Alternatively and preferably, recombinant viral vectors may be engineered, including those derived from viruses such as, but not limited to, herpesviruses, retroviruses, vaccinia viruses, poxviruses, adenoviruses, lentiviruses, adeno-associated viruses, or bovine papilloma viruses.

[0061] In a preferred embodiment, the label utilized in the present invention, associated with the tag used to mark or "paint" endogenous and synthetic chromosomes, is an expressed fluorescent protein; thus, an expression cassette may be used. Expression vectors provide transcriptional and translational regulatory sequences and may provide inducible or constitutive expression when the coding region is operably linked under the transcriptional control of a transcriptional initiation region and a transcriptional and translational termination region. These regulatory regions may be native to the gene encoding the polypeptide or may be derived from an exogenous source, including species-specific endogenous promoters. In general, transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosome binding sites, transcriptional initiation and termination sequences, translational initiation and termination sequences, and enhancer or activator sequences. In addition to constitutive and inducible promoters, strong promoters (e.g., T7, CMV, etc.) are used in the constructs described herein, especially when high expression levels are desired in in vivo (cell systems) or in vitro expression systems. Other exemplary promoters include the mouse mammary tumor virus (MMTV) promoter, the Rous sarcoma virus (RSV) promoter, adenovirus promoters, promoters from the immediate early gene of human CMV, and promoters from the long terminal repeat (LTR) of RSV. Alternatively, the promoter can be provided by, for example, the 5'UTR of a retrovirus.

[0062] Expression vectors generally have convenient restriction sites located near the promoter sequence to allow for the insertion of a nucleic acid sequence encoding a protein of interest. A selectable marker that operates in the expression host may be present to facilitate the selection of cells that contain the vector. In addition, the expression construct may contain additional elements. For example, the expression vector may have one or two replication systems; thus allowing the vector to be maintained in an organism, for example, in mammalian cells for expression and in prokaryotic hosts for cloning and amplification. In addition, the expression construct may contain a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.

[0063] <Production of synthetic chromosomes> The real-time monitoring of the synthetic chromosome production method of the present invention is applicable to all synthetic chromosome production methods currently in use. As briefly discussed above, the real-time monitoring method of the present invention is applicable to all of the "top-down", "bottom-up", minichromosome genetic engineering, and artificial de novo chromosome generation methods used in the art. The "bottom-up" method of synthetic chromosome formation relies on cell-mediated de novo chromosome formation following transfection of permissive cell lines with cloned alpha-satellite sequences comprising centromeres and selectable marker genes appropriate for a typical host cell, with or without telomere and genomic DNA. (For protocols and detailed descriptions of these methods, see, e.g., Harrington et al., Nat. Genet. 15:345-55, 1997; Ikeno et al., Nat. Biotechnol. 16:431-39, 1998; Masumoto et al., Chromosoma 107:406-16, 1998; Ebersole et al., Hum. Mol. Genet. 9:1623-31, 2000; Henning et al., Proceedings of the National Academy of Sciences of the United States of America, 2003). USA 96:592-97 (1999); Grimes et al., EMBO Rep. 2:910-14 (2001); Mejia et al., Genomics 79:297-304 (2002); and Grimes et al., Mol. Ther. 5:798-805 (2002).) Both synthetic and naturally occurring α-satellite repeat arrays cloned into yeast artificial chromosome, bacterial artificial chromosome, or P1-derived artificial chromosome vectors have been used in the art to generate de novo synthetic chromosomes.The products of bottom-up assembly can be linear or circular, comprise simplified and / or concatemerized input DNA with an α-satellite DNA-based centromere, and can typically range in size from 1 to 10 Mb. The bottom-up derived synthetic chromosomes are further engineered to incorporate nucleic acid sequences that allow site-specific incorporation of target DNA sequences onto the synthetic chromosome.

[0064] "Top-down" methods of producing synthetic chromosomes involve sequential random and / or targeted shortening of existing chromosome arms to generate reduced synthetic chromosomes that contain centromeres, telomeres, and origins of DNA replication. (For protocols and detailed descriptions of these methods, see, e.g., Heller et al., Proceedings of the National Academy of Sciences, 1996, 93:7125-30; Saffery et al., Proceedings of the National Academy of Sciences, 2001, 98:5705-10; Choo, Trends in Molecular Medicine, 2003, 111-115). Mol. Med. 2001, 7, 235-37; Barnett et al., Nuc. Ac. Res. 1993, 21, 27-36; Farr et al., Proceedings of the National Academy of Sciences 1991, 88, 7006-10; and Katoh et al., Biochem. Biophys. Res. Commun. 2004, 321, 280-90.) "Top-down" synthetic chromosomes are optimally constructed to lack naturally occurring expressed genes and are engineered to contain DNA sequences that allow site-specific integration of target DNA sequences onto the truncated chromosome, e.g., via a site-specific DNA integrase.

[0065] A third method for producing synthetic chromosomes known in the art is the genetic engineering of naturally occurring minichromosomes, which typically involves radiation-induced fragmentation of a chromosome that contains a functional, e.g., human neocentromere with centromeric function, but lacks α-satellite DNA sequences, and has been genetically engineered to lack nonessential DNA. (For protocols and detailed descriptions of these methods, see, e.g., Auriche et al., Embo Reports 2:102-7, 2001; Moralli et al., Cytogenet. Cell Genet. 94:113-20, 2001; and Carine et al., Somat. Cell Mol. Genet. 15:445-460, 1989.) As with other methods for generating synthetic chromosomes, engineered minichromosomes can be engineered to contain DNA sequences that allow for site-specific integration of target DNA sequences.

[0066] A fourth approach for the production of synthetic chromosomes involves artificial de novo chromosome generation by targeted amplification of specific chromosomal segments. This approach involves large-scale amplification of pericentric / ribosomal DNA regions located on endotic chromosomes. Amplification is triggered by co-transfection of excess DNA specific for the pericentric region of the chromosome, such as ribosomal RNA, together with DNA sequences that allow site-specific integration of the target DNA sequence and also a drug selection marker that is integrated into the pericentric region of the chromosome. (For protocols and detailed descriptions of these methods, see, for example, Csonka et al., J. Cell Science 1999, 144:131-132, 2002). Sci. 2002, 113, 3207-16; Hadlaczky et al., Curr. Opini. Mol. Ther. 2001, 3, 125-32; and Lindenbaum and Perkins et al., Nuc. Ac. Res. 2004, 32, 21, e172.) During this process, targeting of the pericentric regions of end-attached chromosomes with co-transfected DNA leads to extensive chromosomal DNA amplification, duplication / activation of centromeric sequences, and subsequent cleavage and dissociation of the dicentric chromosome to generate a synthetic chromosome based on "break-off" satellite DNA that contains multiple site-specific integration sites. One exemplary embodiment of this process is shown in FIG.

[0067] Figure 5 is a schematic representation of one embodiment of a method for real-time monitoring of chromosome production. Exemplary components are listed and as follows: histone H2B red fluorescent protein tag, which is specific to the endogenous chromosome of human engineered cell line; LacI green fluorescent protein tag, which is specific to synthetic chromosome; pHurDNA vector and pHUPattPPurolacO vector, which deliver components for making synthetic chromosome; LacI green fluorescent protein bound to lacO sequence on synthetic chromosome; and histone H2B red fluorescent protein bound to chromosome. In the process, human engineered cell line is transfected with two vectors that deliver components for making synthetic chromosome, and these vectors are integrated into endogenous chromosome of human engineered cell line. Large-scale amplification of lacO array is triggered, and dicentric chromosome is formed from endogenous chromosome and synthetic chromosome components. In addition, LacI green fluorescent protein tag is localized to lacO array on synthetic chromosome. After mitotic cleavage of the dicentric chromosome, the newly formed synthetic chromosome is maintained in human engineered cell lines. Colocalization of the histone red fluorescent protein tag and the LacI green fluorescent tag allows purification of the synthetic chromosome by dual-color flow cytometry.

[0068] 6 shows simplified construction of an endogenous chromosome-specific tag vector, a synthetic chromosome-specific tag vector, and a hybrid vector specific for endogenous and synthetic chromosomes. Exemplary components are listed and noted below: an endogenous chromosome-specific tag; an endogenous chromosome-binding moiety (e.g., a histone-binding moiety); a first reporter moiety (e.g., a first fluorescent protein); a synthetic chromosome-specific tag; a synthetic chromosome-binding moiety (e.g., a lac repressor); a second reporter moiety (e.g., a second fluorescent protein); and a selection marker (e.g., a drug resistance gene, such as for hygromycin resistance).

[0069] Delivery of components to synthetic chromosome producing cells Vectors carrying the reporter tags of the invention and / or components suitable for synthetic chromosome production can be delivered by any method known in the art to cells for genetic engineering and / or production of synthetic chromosomes. The terms transfection and transformation refer to the uptake of exogenous nucleic acid, e.g., expression vectors, by a host cell, regardless of whether any coding sequences are actually expressed. Numerous methods of transfection are known, e.g., Agrobacterium-mediated transformation, protoplast transformation (including polyethylene glycol (PEG)-mediated transformation, electroporation, protoplast fusion, and microcell fusion), lipid-mediated delivery, liposomes, electroporation, sonoporation, microinjection, particle bombardment, and silicon carbide whisker-mediated transformation, as well as combinations thereof (see, e.g., Paszkowski et al., EMBO Journal, 2003). J., 1984, vol. 3, pp. 2717-2722; Potrykus et al., Mol. Gen. Genet., 1985, vol. 199, pp. 169-177; Reich et al., Biotechnology, 1986, vol. 4, pp. 1001-1004; Klein et al., Nature, 1987, vol. 327, pp. 70-73; U.S. Pat. No. 6,143,949; Paszkowski et al., Cell Culture and Somatic Cell Genetics of Plants, vol. 1, pp. 2717-2722; "Molecular Biology of Plant Nuclear Genes," in Volume 6 of the Journal of Plant Biology, Schell and Vasil, eds., Academic Publishers, 1989; and Frame et al., Plant J.6, pp. 941-948 (1994); direct uptake using calcium phosphate (Wigler et al., Proceedings of the National Academy of Sciences, Vol. 76, pp. 1373-1376 (1979); polyethylene glycol (PEG)-mediated DNA uptake; lipofection (see, e.g., Strauss, Meth. Mol. Biol., Vol. 54, pp. 307-327 (1996)); microcell fusion (Lambert, Proceedings of the National Academy of Sciences, Vol. 88, pp. 5907-5911 (1991); U.S. Patent No. 5,396,767; Sawford et al., Somatic Cell and Molecular Genetics, Vol. 1, pp. 1372-1375 (1996)); Mol. Genet. 13:279-284 (1987); Dhar et al., Somatic Cell and Molecular Genetics 10:547-559 (1984); and McNeill-Killary et al., Meth. Enzymol. 254:133-152 (1995); lipid-mediated Delivery systems (e.g., Teifel et al., Biotechniques, 1995, vol. 19, pp. 79-80; Albrecht et al., Ann. Hematol., 1996, vol. 72, pp. 73-79; Holmen et al., In Vitro Cellular and Developmental Biology, Animals, 1997, vol. 10, pp. 111-115; Vitro Cell Dev. Biol. Anim., 1995, vol. 31, pp. 347-351; Remy et al., Bioconjug. Chem., 1994, vol. 5, pp. 647-654; Le Bolch et al., Tetrahedron Lett., 1995, vol. 36, pp. 6681-6684; and Loeffler et al., Meth. Enzymol., 1993, vol. 217, pp. 2171-2176.599-618); or by other suitable methods known to those of skill in the art. Methods for delivery of synthetic chromosomes are further described in U.S. Patent Application Serial No. 09 / 815,979. Successful transfection is generally recognized by detection of the presence of the heterologous nucleic acid in the transfected cells, such as any visualization of the heterologous nucleic acid, expression of a selectable marker, or any indication of the operation of the vector within the host cell. For a description of delivery methods useful in carrying out the present invention, see U.S. Pat. Nos. 5,011,776; 5,747,308; 4,966,843; 5,627,059; 5,681,713; Kim and Eberwine, Anal. Bioanal. Chem., 2010, Vol. 397, No. 8, pp. 3173-3178.

[0070] <<Tag with Label>> As discussed above in the discussion of Figures 1-4, the present invention relates to two tags: one labeled tag specific to an endogenous chromosome of the synthetic chromosome producing cell line, and one differentially-labeled tag specific to a sequence on the synthetic chromosome to be produced. However, in further embodiments, it is contemplated that additional tags are contemplated to identify multiple regions of the synthetic chromosome. The labeled tag specific to the endogenous chromosome of the synthetic chromosome producing reporter cell line is preferably a tag specific to all endogenous chromosomes of the cell line, for example, a histone-specific marker, such as a marker specific for histones H1, H2A, H2B, H3, H4, and H5. Alternatively, it is contemplated that a population of tags specific to different chromosomes, all linked to a single label (e.g., a fluorescent protein), may be used. In a preferred embodiment, the endogenous chromosome-specific labeled tag is a fusion peptide; however, it is also contemplated that the endogenous chromosome-specific labeled tag may also be a nucleic acid / protein chimera with a tag moiety that is a nucleic acid sequence specific for the endogenous chromosome and a label moiety that is a protein, such as a fluorescent or phosphorescent protein, or the tag moiety is a TALEN protein specific for the endogenous chromosome and with a label moiety that is a protein, such as a fluorescent, phosphorescent, or luminescent protein. In addition, the tag moiety may also be a modified CRISPR moiety (nuclease deficient) fused to a fluorescent protein and co-expressing an RNA moiety that provides specific binding and visualization of the synthetic chromosome (see, e.g., Chen et al., Cell 2013, vol. 155, pp. 1479-1491; and Chen and Huang, Methods in Enzymolgy 2014, vol. 546, pp. 337-354). Figures 8-10 are schematic representations of the use of the CRISPR / Cas fluorescent visualization system to monitor, isolate, and / or purify synthetic chromosomes.In Figure 8, a single reporter system is used in which the CRISPR components are fused to a fluorescent protein and the co-expressed RNA components provide specific binding and visualization of the synthetic chromosome. Figure 9 shows two different cell lines, one containing one CRISPR / Cas / fluorescence component bound to a synthetic chromosome, and the other cell line shows two CRISPR / Cas / fluorescence components bound to two synthetic chromosomes, one with fluorescent protein 1 and one with fluorescent protein 2. Figure 10 shows the use of two different CRISPR / Cas / fluorescence components to detect site-specific arm exchange between the endogenous, i.e., chromosome native to the cell producing the synthetic chromosome, and the synthetic chromosome. The two different CRISPR / Cas / fluorescence components also allow the synthetic chromosome to be isolated.

[0071] Furthermore, in a preferred embodiment, labeled tags are chosen that are specific to and will mark all endogenous chromosomes; however, as mentioned, it should be clear to one skilled in the art that it is also contemplated that more than one labeled tag may be used, such as multiple labeled tags specific to a single or a few chromosomes, or multiple tags each specific to a single chromosome.

[0072] In a preferred embodiment, the synthetic chromosome-specific tag is a unique nucleic acid sequence on the synthetic chromosome, e.g., any of the four n The synthetic chromosome-specific tag comprises a nucleic acid sequence complementary to a list of nucleotides (that are likely to be unique and can be screened against a database of known sequences) and a fluorescent protein as a nucleic acid / protein chimera. In a preferred embodiment, the synthetic chromosome-specific tag is specific to a site-specific incorporation sequence (acceptor site) on the synthetic chromosome; thus, the signal from the labeled tag serves indirectly as a measure of the number of acceptor sites, and the synthetic chromosomes produced can be classified by the number of acceptor sites on the chromosome.

[0073] Labels contemplated for use in the present invention include any visible label that can be used in conjunction with tags specific to endogenous chromosomes of synthetic chromosome producing cell lines and tags specific to sequences on synthetic chromosomes. In a preferred embodiment, the label is a fluorescent label that is transcribed and translated by the synthetic chromosome producing cells into a fusion protein or nucleic acid / protein chimera incorporating the tag. Fluorescent proteins that are particularly useful in the present invention include, but are not limited to, TagBFP, TagCFP, TagGFP2, TagYFP, TagRFP, FusionRed, mKate2, TurboGFP, TurboYFP, TurboRFP, TurboFP602, TurboFP635, or TurboFP650 (all available from Evrogen, Moscow, Russian Federation); AmCyan1, AcvGFP1, ZsGreen1, ZsYellow1, mBanana, mOrange, mOrange2, DsRed-Express2, DsRed-Express3, DsRed-Express4, DsRed-Express5, DsRed-Express6, DsRed-Express7, DsRed-Express8, DsRed-Express9, DsRed-Express10, DsRed-Express11, DsRed-Express12, DsRed-Express13, DsRed-Express14, DsRed-Express15, DsRed-Express16, DsRed-Express17, DsRed-Express18, DsRed-Express19, DsRed-Express20, DsRed-Express21, DsRed-Express22, DsRed-Express23, DsRed-Express24, DsRed-Express25, DsRed-Express26, DsRed-Express27, DsRed-Express28, ​​DsRed-Express39, DsRed-Express31, DsRed-Express32, DsRed-Express33, DsRed-Express34, DsRed Examples of fluorescent tags include s, tdTomato, DsRed-Monomer, DsRed2, AsRed2, mStrawberry, mCherry, HcRed1, mRaspberry, E2-Crimson, mPlum, Dendra2, Timer, and PAmCherry (all available from Clontech, Palo Alto, Calif.); HALO-tag®; infrared (near-infrared shifted) tag (available from Promega, Madison, Wis.); and other fluorescent tags known in the art, as well as those later discovered.

[0074] Visualization and Monitoring When the reporter tag of the present invention is labeled with a fluorescent label, the localization of the label in the synthetic chromosome-producing cells can be performed with a fluorescent microscope. Generally, the cells are excited with a light source at the excitation wavelength of the particular fluorescent label used, and the resulting fluorescence at the emission wavelength is detected. In a preferred embodiment, the excitation light source is a laser suitable for excitation of the fluorescent label. The confocal microscope used to detect the label can be automated with a computer-controlled stage to automatically scan the entire cell culture dish. Similarly, the microscope can be equipped with a phototransducer (e.g., photomultiplier tube, solid-state array, CCD camera, etc.) attached to an automated data acquisition system to automatically record the fluorescent signal generated by each cell or cell colony in the culture. Alternatively, an imaging flow cytometer, such as Flowsight® (Amnis, Seattle, WA, USA), can be used to capture an image of each "cell" in the flow stream following excitation with a laser; thus automating the evaluation of each cell colony for synthetic chromosome production.

[0075] Lindenbaum and Perkins et al., Nucleic Acid Research, 2004, vol. 32, no. 21, :e172, describe the production of an artificial chromosome expression (ACE) system using mammalian satellite DNA using prior art techniques. In this prior art system, conventional single-color and dual-color FISH analysis and high-resolution FISH were performed using PCR-generated or nick-translated probes. For detection of telomeric sequences, mitotic spreads were hybridized with commercially available peptide nucleic acid probes. Microscopy was performed using a fluorescent microscope. The process took approximately three months. The present invention allows the repeated FISH analysis to be reduced or completely eliminated, the new method of the present application eliminates the need for potentially mutagenic dyes that would have been used to sort chromosomes, and allows the development of synthetic chromosomes to be observed in real time. In addition, the methods of the present invention allow for easier genetic manipulation downstream of a synthetic chromosome when a DNA element of interest is inserted into the synthetic chromosome. Figure 7 shows a timeline for the method of producing a synthetic chromosome as described herein. EXAMPLES

[0076] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments described below are all or the only experiments performed. Those skilled in the art will appreciate that numerous changes and / or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive.

[0077] Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0078] Example 1: Construction of a vector for delivering tags For real-time monitoring of synthetic platform chromosomes, the cell line HT1080 was engineered to carry an endogenous chromosome-specific tag vector (a tag that identifies all naturally occurring endogenous chromosomes) along with a synthetic chromosome-specific tag vector (a tag that identifies the synthetic chromosome separately from the endogenous chromosomes) (see FIG. 6; see also, e.g., Robinett et al., J Cell Biol. 1996, vol. 135(6): pp. 1685-700). Endogenous chromosome-specific tags can be constructed using chromosome-specific binding moieties, e.g., DNA-binding proteins such as histone proteins (see, e.g., Kimura et al., J Cell Biol. 2000, vol. 153, no. 7:1341-53), proteins involved in chromatin structures, or genome editing proteins, e.g., zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) (see, e.g., Thanisch et al., Nucleic Acids Res. 2014, vol. 42, no. 6:e38; and Miyanari et al., Nat Struct Mol. Biology 2013, vol. 11, no. 11:11-12; and Miyanari et al., Nat Struct Mol. Biology 2013, vol. 11, no. 11:11-12). Biol. 2013, vol. 20, no. 11: 1321-24), proteins of the CRISPER / Cas system (see, e.g., Anton et al., Nucleus 2014, vol. 5, no. 2: 163-72), or artificial meganucleases. For endogenous chromosome-specific tags, the chromosome-specific binding moiety was fused and expressed together with a reporter moiety that was detected by standard microscopy or flow cytometry methodologies. The reporter moiety in a preferred embodiment comprises a protein fusion tag (e.g., HaloTag) linked to a molecule such as a fluorescent molecule (e.g., green fluorescent protein) or a chemical ligand.

[0079] The synthetic chromosome-specific tag vectors comprised DNA sequence-specific binding moieties, such as bacterial Lac repressor proteins that bind to engineered Lac operon sequences on synthetic chromosomes, or genome editing proteins (e.g., TALENS, RNA / CRISPR-CAS) that were engineered to bind to DNA sequences specific to synthetic chromosomes. The synthetic chromosome-specific tags were fused and expressed with reporter moieties that could be detected by standard microscopy or flow cytometry methodologies. The reporter moieties fused to the synthetic chromosome-specific tags, in a preferred embodiment, comprise protein fusion tags (e.g., HaloTag®) that are linked to molecules such as fluorescent molecules (e.g., red fluorescent protein) or chemical ligands.

[0080] The endogenous and synthetic chromosome-specific tags are expressed from a promoter (e.g., the human glucose-6-phosphate isomerase promoter) and expressed from two unique vectors, although the endogenous and synthetic chromosome-specific tags could be combined on a single vector with appropriate drug selection (e.g., an expression gene conferring hygromycin resistance) (see again FIG. 5). Alternatively, the endogenous and synthetic chromosome-specific tags could be expressed from an inducible promoter (e.g., a tetracycline-regulatable promoter). For subsequent removal of the tag and / or tag vector sequences from the engineered cell line, site-specific recombination sequences (e.g., loxP sequences) were flanked on both sides of the endogenous and synthetic chromosome-specific tag vectors, and these sequences were selectively removed by introducing the appropriate site-specific recombinase (e.g., Cre protein) and identifying the resulting recombinants by standard microscopy or flow cytometry methodologies.

[0081] Example 2: Delivery of tag vector into host cell line genome For transfection of the tag vector into the host cell line genome, linearized tag vector DNA was introduced into the HT1080 host cell genome using standard mammalian cell transfection reagents such as, but not limited to, lipofectamine® LTX (ThermoFisher) or Viafect™ (Promega) and grown in culture for approximately 2-5 days. Once integrated into the host cell genome, expression from the integrated endogenous and synthetic chromosome-specific tags allowed for FACs selection of host cells producing both tags.

[0082] The day before transfection, cells of the HT1080 host cell line were plated at approximately 2–8 × 10 cells per well of a 24-well tissue culture dish. 4The cells were distributed to a density of 1000 cells / ml and a tagged element vector such as pMC1 was purified (e.g., using the Qiagen EndoFree® Plasmid Maxi Kit, Qiagen, Valencia, CA, USA), linearized, and the concentration of the vector was determined for transfection. Cultured HT1080 cells were fed 3-5 hours before transfection. The pMC1 vector comprises the EF1 / HTLV promoter, green fluorescent protein marker sequence, lacIF; SV40 polyA, IFN-beta scaffold / matrix attachment region, R6K origin of replication, beta glow matrix attachment region, histone H2B sequence, mCherry selection marker, IRES sequence, hygromycin resistance gene, bGH polyA, and 3'UTR sequence. 250-500 ng of vector DNA per well of a 24-well subconfluent tissue culture dish was used to transfect HT1080 cells using, for example, Lipofectamine®-LTX-mediated transfection (Life Technologies, Grand Island, NY, USA). Cells were maintained for 1-5 days after transfection, at which point they were trypsinized and transferred to 10 cm dishes. At the time of plating into the 10 cm dishes or 1-3 days after plating, hygromycin selection medium was added to the 10 cm dishes. Hygromycin-resistant clones were ring cloned and grown in 24-well dishes. Clones expressing both tag elements were single-cell sorted into 96-well tissue culture dishes. Single cell clones were expanded in culture and monitored using fluorescence microscopy as they grew in culture to identify clones with optimal performance of the incorporated tag elements. The best candidates were expanded in culture and cryopreserved using standard methods for future use.

[0083] Example 3: De novo generation of artificial chromosomes based on satellite DNA For de novo production of synthetic chromosomes, exogenous DNA sequences were introduced into the HT1080 synthetic chromosome production cell line and, upon integration into the heterochromatic region flanking the centromere of the acrototic chromosome, triggered extensive amplification of the short arm of the acrototic chromosome (the rDNA / centromere region). During the amplification event, the centromere was duplicated to generate a dicentric chromosome with two active centromeres. Subsequent mitotic events resulted in cleavage and dissociation of the dicentric chromosome, resulting in the release of a fragment approximately 20-120 Mb in size composed primarily of satellite repeat sequences with co-amplified subdomains of the transfected transgene that may contain copies of amplified rDNA. The newly generated synthetic chromosomes were authenticated by observing fluorescent staining of the chromosomes via the endogenous chromosome tags and the synthetic chromosome tags that had been engineered into the HT1080 synthetic chromosome production cell line.

[0084] The day before transfection, cells of the HT1080 synthetic chromosome production cell line were cultured at approximately 2.0–8.0 × 10 4The cells were distributed into 24-well tissue culture dishes at a density of 1000 cells / well adherent cells, and the vectors comprising the exogenous DNA were purified (e.g., using Qiagen EndoFree® Plasmid Maxi Kit), linearized, and the concentration of the vector was determined for transfection. The cultured HT1080 cells were fed 3-5 hours before transfection. 225 ng of pSPOP48lacHur DNA vector and 12.5 ng of EF1alphaattPPuro vector per 24-well subconfluent tissue culture dish were used to transfect HT1080 cells using standard transfection reagents, such as ThermoFisher's Lipofectamine® LTX, Promega's Viafect™, or Invitrogen's calcium phosphate transfection kit. The pSPOP48lacHur DNA vector comprises LacI repeat sequences and ribosomal DNA sequences. The EF1alphaattPPuro vector comprises components for a site-specific recombination system as well as ampicillin and puromycin resistance genes. Cells were maintained for 1-3 days after transfection, at which point they were trypsinized and replated in 10 cm dishes. Selection medium was added to the 10 cm dishes at the time of plating or 1-3 days after plating. Selective conditions were maintained for 10-21 days with medium changes every 2-3 days. Antibiotic-resistant clones were picked when colonies reached a diameter of 2-3 mm. Well-isolated colonies are preferred. Cells were lifted by use of a cloning cylinder and trypsin and transferred to 24-well plates for amplification.

[0085] Example 4: Real-time monitoring of synthetic chromosome platforms For the generation of synthetic chromosome platforms, HT1080 synthetic chromosome-producing reporter cells expressing an endogenous chromosome-specific tag, such as human H2B-RFP fusion protein, and a synthetic chromosome-specific tag, such as LacI-GFP, a Lac repressor fused to GFP, were plated at approximately 2.0–8 × 10 cells / well in 24-well tissue culture dishes. 4The cells were seeded to yield 100 adherent cells. The chromosome-specific tag protein is constitutively expressed. One day after seeding, transfection is performed using standard transfection methodologies, such as Invitrogen's calcium phosphate transfection kit, Lipofectamine®-LTX-mediated transfection (Life Technologies, Inc., Grand Island, NY, USA) or Viafect™ (Promega). For transfection into HT1080 engineered cell lines, the linearized vector (pSPOP48lacHurDNA) is co-transfected with a linearized plasmid (pEF1alphaattPPuro) carrying a site-specific recombination site (e.g., attP) inserted between the human promoter and a drug selection marker (e.g., puromycin resistance). In addition, pSPOP48lacHurDNA contains approximately 48 copies of a synthetic chromosome-specific tag recognition sequence (e.g., 48 copies of the lac operator sequence, lacO), i.e., the sequence recognized and bound by the synthetic chromosome-specific tag LacI-GFP. For co-transfection, an excess of pSPOP48lacHurDNA is delivered along with the pEF1alphaattPPuro plasmid (>3:1 molar excess of pSPOP48lacHurDNA over pEF1alphaattPPuro). 24 hours after transfection, HT1080 cells from each well of a 24-well dish are trypsinized, plated into 10 cm cell culture dishes (one well of a 24-well dish per 10 cm dish), and incubated. One to four days after transfection, the medium is replaced with selection medium consisting of complete medium containing 0.5 micrograms / ml puromycin. Selection medium is changed three times a week for 2 to 3 weeks until the emergence of drug-resistant clones is visible.Note that if initial fluorescence microscopy of candidates in 24-well dishes reveals clones with mature synthetic chromosome break-off products, then examination of cells by fluorescence microscopy can be shifted to earlier observation in 10 cm tissue culture dishes prior to ring cloning. Because the process of generating synthetic chromosomes has been ad hoc thus far, the time for the formation of the first synthetic chromosomes is not fully determined.

[0086] When clones are visible, 96 clones are isolated by ring cloning, and each clone is transferred into a well of a 24-well tissue culture dish and cultured with drug selection. At this time, near-confluent cells in the 24-well dish (2-10 days) are harvested using trypsinization, and individual clonal cell suspensions are distributed into identically positioned wells of two separate 24-well plates. One dish is used for real-time monitoring of synthetic chromosome production, while the other equivalent dish is used for routine maintenance and growth. During growth in the 24-well dishes monitored for synthetic chromosome production, cells are analyzed every 48 hours using standard fluorescence microscopy and confirmed for the appearance of mitotic cells showing red-labeled endogenous chromosomes (presence of H2B-RFP bound to endogenous chromosomes) and co-localized punctate staining of the synthetic chromosome tag; i.e., the presence of LacI-GFP tag on newly synthesized platform chromosomes or on the "sausage" chromosomes, which are precursors of the platform chromosomes to be synthesized. Targeting of the pSPOP48lacHurDNA and pEF1alphaattPPuro vectors into the centromere / rDNA regions of native acrotome chromosomes results in endogenous large-scale amplification of the incorporated plasmids, including proliferation of lacO-repeat repeats, and the formation of dicentric chromosomes followed by abscission and synthetic chromosome production. If necessary, a cell cycle arresting agent such as colcemid (KaryoMax® colcemid solution, Life Technologies, Grand Island, NY, USA) can be added to increase the population of cells in the G2 / M phase to facilitate visual inspection of condensed chromosomes in real time. Real-time monitoring of synthetic chromosome production utilizing endogenous and synthetic chromosome tags alleviates the need to monitor production using static methods such as fluorescence in situ hybridization (FISH).When the cells from the 24-well dish used for routine maintenance and expansion reach near confluence, they are subsequently passaged into two separate 24-well dishes, one for further expansion and maintenance and the other for real-time monitoring until clones containing the newly produced, stably mitotic synthetic chromosomes are identified (approximately 2-4 weeks).Photomicrographs and / or videos of the cultures throughout the process are taken to document the progression of synthetic chromosome formation, i.e., incorporation of the exogenous DNA element; amplification of the chromosomal region with the integrated exogenous DNA; formation of a dicentric chromosome; and mitotic cleavage of the dicentric chromosome.

[0087] Isolated clones containing the new synthetic chromosomes can be further expanded into three 15 cm dishes; one dish is reserved for long-term cryogenic storage; the remaining two dishes are metaphase blocked using colcemid, and the synthetic chromosomes are harvested and purified as previously described (see, e.g., Vanderbyl et al., Cytometry 44(2):100-05, 2001; and Lindenbaum and Perkins et al., Nucleic Acid Research 32(21):e172, 2004). In contrast to previous methods that utilize potential mutagens (e.g., Hoechst® and chromomycin A3) to counterstain the chromosomes, the binding and presence of H2B-RFP and LacI-GFP on the synthetic platform chromosomes allows for dual-color fluorescence-activated sorting and subsequent isolation of the synthetic chromosomes for delivery to cells of interest. Furthermore, the isolated synthetic chromosomes coated with H2B-RFP and LacI-GFP can then be used to evaluate and optimize the delivery of the synthetic chromosomes to the cell type of interest, i.e., to fluorescently monitor the percentage of transfected cells that have the delivered synthetic chromosomes. An overview of this process is shown in FIG. 7.

[0088] The preceding description merely illustrates the principles of the present invention. Of course, those skilled in the art will be able to devise various arrangements that embody the principles of the present invention and are within the spirit and scope of the present invention, although not explicitly described or shown herein. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the present invention and the concepts contributed by the inventor to advance the art, and should be construed as without limitation to such specifically recited examples and conditions. Furthermore, all descriptions herein reciting principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. In addition, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Thus, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims. In the following claims, unless the term "means" is used, no feature or element recited therein is to be construed as a means-plus-function limitation pursuant to 35 U.S.C. 35 U.S.C. 112, sixth paragraph.

[0089] The technical ideas that can be understood from the above-described embodiment will be described below as supplementary notes. [Appendix 1] 1. A method for screening for the production of a synthetic chromosome, the method comprising: Providing a synthetic chromosome-producing reporter cell line; transfecting a synthetic chromosome-producing reporter cell line with the endogenous chromosome tag and the synthetic chromosome tag; transfecting a synthetic chromosome production reporter cell line with synthetic chromosome production constructs; monitoring the production of the synthetic chromosome in the synthetic chromosome-producing reporter cell line by using the endogenous chromosome tag and the synthetic chromosome tag.

[0090] [Appendix 2] The method of claim 1, wherein the endogenous chromosomal tag and the synthetic chromosomal tag are stably integrated into the genome of a synthetic chromosome-producing reporter cell line.

[0091] [Appendix 3] The method of claim 1, wherein the endogenous chromosomal tag becomes stably integrated into the genome of the synthetic chromosome producing reporter cell line and the synthetic chromosomal tag becomes stably integrated into the synthetic chromosome.

[0092] [Appendix 4] The method of claim 1, wherein the endogenous chromosomal tag and the synthetic chromosomal tag are stably incorporated into a synthetic chromosome.

[0093] [Appendix 5] The method of claim 1, wherein the endogenous chromosomal tag becomes stably integrated into the synthetic chromosome and the synthetic chromosomal tag becomes stably integrated into the genome of the synthetic chromosome-producing reporter cell line.

[0094] [Appendix 6] 6. The method of any one of claims 1 to 5, wherein the arms of endogenous chromosomes present in the synthetic chromosome-producing reporter cell line comprise recombination sites compatible for interaction with the recombination sites in the synthetic chromosome.

[0095] [Appendix 7] 6. The method of any one of claims 1 to 5, wherein the endogenous chromosomal tag and the synthetic chromosomal tag comprise fluorescent tags, and the monitoring step is performed by fluorescence microscopy.

[0096] [Appendix 8] 8. The method of claim 7, wherein the fluorescent tag is selected from TagBFP, TagCFP, TagGFP2, TagYFP, TagRFP, FusionRed, mKate2, TurboGFP, TurboYFP, TurboRFP, TurboFP602, TurboFP635, TurboFP650, AmCyan1, AcvGFP1, ZsGreen1, ZsYellow1, mBanana, mOrange, mOrange2, DsRed-Express2, DsRed-Express, tdTomato, DsRed-Monomer, DsRed2, AsRed2, mStrawberry, mCherry, HcRed1, mRaspberry, E2-Crimson, mPlum, Dendra2, Timer, and PAmCherry, HALO-tag, or infrared-shifted fluorescent protein.

[0097] [Appendix 9] The method according to any one of claims 1 to 5, wherein the tags of the endogenous chromosomal tag and the synthetic chromosomal tag comprise chemiluminescent tags and the monitoring step is carried out by chemiluminescence microscopy, or comprise phosphorescent tags and the monitoring step is carried out by phosphorescence microscopy.

[0098] [Appendix 10] 6. The method of any one of claims 1 to 5, wherein the endogenous chromosomal tag comprises a marker specific for histone H1, H2A, H2B, H3, H4, or H5.

[0099] [Appendix 11] The synthetic chromosomal tags were screened against a database of known sequences. n 6. The method according to any one of claims 1 to 5, comprising a nucleotide.

[0100] [Appendix 12] 6. The method according to any one of appendix 1 to 5, wherein the synthetic chromosome-producing reporter cell line is selected from a mammalian cell line, an embryonic cell line, a pluripotent cell line, an adult-derived stem cell, a reprogrammed cell line or a human cell line.

[0101] [Appendix 13] 13. The method of claim 12, wherein the synthetic chromosome-producing reporter cell line is the human cell line HT1080.

[0102] [Appendix 14] 6. The method of any one of claims 1 to 5, wherein the synthetic chromosome production reporter cell line is transfected with synthetic chromosome production components to produce synthetic chromosomes by a top-down approach.

[0103] [Appendix 15] 6. The method of any one of claims 1 to 5, wherein the synthetic chromosome production reporter cell line is transfected with synthetic chromosome production components to produce synthetic chromosomes by a bottom-up approach.

[0104] [Appendix 16] 6. The method of any one of claims 1 to 5, wherein the synthetic chromosome production reporter cell line is transfected with synthetic chromosome production components to produce synthetic chromosomes by genetic engineering of naturally occurring minichromosomes.

[0105] [Appendix 17] The method of any one of claims 1 to 5, wherein the synthetic chromosome production reporter cell line is transfected with synthetic chromosome production components to produce synthetic chromosomes by de novo chromosome generation through targeted amplification of chromosome segments.

[0106] [Appendix 18] 18. The method of claim 17, wherein the chromosome segment is a pericentric region of a chromosome. [Appendix 19] The method according to any one of claims 1 to 5, wherein the endogenous chromosomal tag comprises a plurality of endogenous chromosomal tags.

[0107] [Appendix 20] The method according to any one of appendix 1 to 5, wherein the endogenous chromosome tag is a fusion protein, a nucleic acid / protein chimera, a nucleic acid / protein complex (e.g., an RNA / CRISPR-CAS9 complex), or a moiety comprising a TALEN protein specific to the endogenous chromosome and a fluorescent or phosphorescent label.

[0108] [Appendix 21] A synthetic chromosome-producing reporter cell line comprising an endogenous chromosome tag and a synthetic chromosome tag, wherein the endogenous chromosome tag and the synthetic chromosome tag are stably integrated into the genome of the synthetic chromosome-producing reporter cell line.

[0109] [Appendix 22] A synthetic chromosome produced by the method according to any one of claims 1 to 20. [Appendix 23] A synthetic chromosome produced by the method of any one of appendix 1 to 20 and having a size of 20 to 120 Mbp.

[0110] [Appendix 24] A synthetic chromosome comprising: comprising sites for attaching at least two differently labeled tags, a first tag detects a first site present on both the synthetic chromosome and the endogenous chromosome; A synthetic chromosome, wherein a tag specific to a second synthetic chromosome identifies a second site present on said synthetic chromosome separate from the endogenous chromosome.

[0111] [Appendix 25] 25. The synthetic chromosome of claim 24, having a size of 20-120 Mbp. [Appendix 26] The second site is a 4-nucleotide sequence that has been screened against a database of known sequences. n 25. The synthetic chromosome of claim 24, comprising a synthetic chromosome-specific sequence of nucleotides.

[0112] [Appendix 27] 25. The synthetic chromosome of claim 24, wherein the first tag detects multiple sites present on both the endogenous chromosome and the synthetic chromosome.

[0113] [Appendix 28] 28. The synthetic chromosome of claim 27, wherein the multiple sites comprise multiple copies of the same site. [Appendix 29] 25. The synthetic chromosome of claim 24, wherein the first tag comprises a domain that binds to at least one histone selected from H1, H2A, H2B, H3, H4 and H5.

[0114] [Appendix 30] 28. The synthetic chromosome of claim 27, wherein the plurality of sites comprises different sites for binding by two or more different tags.

[0115] [Appendix 31] 25. The synthetic chromosome of claim 24, wherein the first tag and / or the second tag comprises a tag selected from a fusion protein, a nucleic acid / protein chimera, a nucleic acid / protein complex, a portion comprising a TALEN protein, and a nuclease-deficient CRISPR portion, and further comprises a fluorescent or phosphorescent label.

[0116] [Appendix 32] 25. A eukaryotic host cell comprising the synthetic chromosome of appendix 24. [Appendix 33] 33. A eukaryotic host cell comprising the synthetic chromosome of claim 32, wherein the first tag and the second tag are expressed from the genome of the eukaryotic host cell.

[0117] [Appendix 34] 33. A eukaryotic host cell comprising the synthetic chromosome of claim 32, wherein the first tag and the second tag are expressed from the synthetic chromosome.

[0118] [Appendix 35] 33. A eukaryotic host cell comprising the synthetic chromosome of claim 32, wherein the first tag is expressed from the genome of the eukaryotic host cell and the second tag is expressed from the synthetic chromosome.

[0119] [Appendix 36] 33. A eukaryotic host cell comprising the synthetic chromosome of claim 32, wherein the first tag is expressed from the synthetic chromosome and the second tag is expressed from the genome of the eukaryotic host cell.

[0120] [Appendix 37] 25. The synthetic chromosome of claim 24, wherein the second site present in the synthetic chromosome comprises multiple copies of the same site.

[0121] [Appendix 38] 38. The synthetic chromosome of claim 37, wherein the second site comprises an array of multiple lac operator (lacO) sequences and the tag specific to the synthetic chromosome comprises LacI.

[0122] [Appendix 39] 29. The synthetic chromosome of claim 29, A synthetic chromosome, wherein the first tag and the second tag are labeled with a fluorescent or phosphorescent label, and the second tag binds to a nucleic acid sequence on the synthetic chromosome.

Claims

1. A synthetic chromosome, the synthetic chromosome comprising: an amplified pericentromeric rDNA region; and sites for binding at least two differently labeled tags, each of which is visualizeable in a living animal host cell; a first site present in both the synthetic chromosome and the endogenous chromosome of the host cell that is recognizable by a tag carrying a first visible label; a second site present on the synthetic chromosome, separate from the endogenous chromosome of the host cell, that is recognizable by a tag carrying a second visible label, and that, when the synthetic chromosome is present in the host cell, a) the first tag and the second tag are expressed from the genome of the host cell; or b) the first tag and the second tag are expressed from the synthetic chromosome; or c) the first tag is expressed from the genome of the host cell and the second tag is expressed from the synthetic chromosome; or d) a synthetic chromosome, wherein the first tag is expressed from the synthetic chromosome and the second tag is expressed from the genome of the host cell.

2. A synthetic chromosome as described in claim 1, having a size of 20 to 120 Mbp.

3. A synthetic chromosome as described in claim 1, wherein the second portion comprises a sequence specific to the synthetic chromosome of 4n nucleotides, the sequence of which has been screened against a database of known sequences.

4. A synthetic chromosome as described in claim 1, wherein the first tag detects multiple sites present in both the endogenous chromosome and the synthetic chromosome.

5. A synthetic chromosome as described in claim 4, wherein the multiple sites include multiple copies of the same site.

6. The synthetic chromosome described in claim 1, wherein the first tag includes a domain that binds to at least one histone selected from H1, H2A, H2B, H3, H4 and H5.

7. A synthetic chromosome as described in claim 4, wherein the multiple sites include different sites for binding by two or more different tags.

8. The synthetic chromosome of claim 1, wherein the first tag and / or the second tag comprises a tag selected from a fusion protein, a nucleic acid / protein chimera, a nucleic acid / protein complex, a moiety comprising a TALEN protein, and a nuclease-deficient CRISPR moiety, and the visible label is fluorescent or phosphorescent.

9. An animal host cell, the animal host cell further comprising a synthetic chromosome stably encoding and expressing at least two differently labeled tags that are visible in a living host cell and comprising sites for binding said at least two differently labeled tags, the at least two differently labeled tags are stably expressed in the living host cell from the host cell genome, from the synthetic chromosome, or a combination thereof; a first site present in both the synthetic chromosome and the endogenous chromosome of the host cell is identified by binding of a first visible labeled tag; An animal host cell, wherein a second site present on the synthetic chromosome but not on an endogenous chromosome of the host cell is identified by binding of a tag specific to the second visibly labeled synthetic chromosome.

10. An animal host cell comprising the synthetic chromosome described in claim 9, wherein the first visibly labeled tag and the second visibly labeled tag are expressed from the genome of the animal host cell.

11. An animal host cell comprising the synthetic chromosome described in claim 9, wherein the tag with the first visible label and the tag with the second visible label are expressed from the synthetic chromosome.

12. An animal host cell comprising the synthetic chromosome described in claim 9, wherein the first visibly labeled tag is expressed from the genome of the animal host cell and the second visibly labeled tag is expressed from the synthetic chromosome.

13. An animal host cell comprising the synthetic chromosome described in claim 9, wherein the first visibly labeled tag is expressed from the synthetic chromosome and the second visibly labeled tag is expressed from the genome of the animal host cell.

14. A synthetic chromosome as described in claim 1, wherein the second site present in the synthetic chromosome includes multiple copies of the same site.

15. The synthetic chromosome described in claim 14, wherein the second site comprises an array of multiple lac operator (lacO) sequences, and the tag specific to the synthetic chromosome comprises LacI.

16. The synthetic chromosome of claim 6, A synthetic chromosome, wherein the first tag and the second tag are labeled with a fluorescent or phosphorescent label, and the second tag binds to a nucleic acid sequence on the synthetic chromosome.

Images