581 results about "Intein" patented technology

The current invention provides regulatory regions from the rice actin 2 gene. In particular, the current invention provides the rice actin 2 promoter and actin 2 intron. Compositions comprising these sequences are described, as well as transformation constructs derived therefrom. Further provided are methods for the expression of transgenes in plants comprising the use of these sequences. The methods of the invention include the direct creation of transgenic plants with the rice actin 2 intron and/or promoter directly by genetic transformation, as well as by plant breeding methods. The actin 2 sequences of the invention represent a valuable new tool for the creation of transgenic plants, preferably having one or more added beneficial characteristics.

DNA constructs comprise a first exon sequence of nucleotides encoding a first peptide or polypeptide, a second exon sequence of nucleotides encoding a second peptide or polypeptide and a third sequence of nucleotides between the first and second sequences encoding a heterologous intron, for example that of Tetrahymena thermophila nuclear pre-rRNA, between RNA splice sites and a site-specific recombination sequence, such as loxP, within the intron, the exons together encoding a product peptide or polypeptide. Such constructs are of use in methods of production of peptides or polypeptides, transcription leading to splicing out of the intron enabling translation of a single chain product peptide or polypeptide. Isolated nucleic acid constructs consisting essentially of a sequence of nucleotides encoding a self-splicing intron with a site-specific recombination sequence within the intron, for use in creation of constructs for expression of peptides or polypeptides, are also provided.

The present invention provides synthetic 5′UTRs comprising a first polynucleotide fragment and a second polynucleotide fragment, wherein the first polynucleotide fragment comprises at least one splice site of a first eukaryotic gene, the second polynucleotide fragment comprises at least a portion of 5′ untranslated region of a second eukaryotic gene, and the first polynucleotide fragment is located 5′ of the second polynucleotide fragment. In one embodiment, the first polynucleotide fragment comprises the second intron of a sarcoplasmic/endoplasmic reticulum calcium ATPase gene and the second polynucleotide fragment comprises at least a portion of the 5′ untranslated region (5′UTR) of a eukaryotic casein gene. The synthetic 5′UTRs are useful for increasing the expression of a transgene when positioned between a promoter and a transgene within an expression vector. The present invention also provides vectors comprising synthetic 5′UTRs and methods for increasing the expression of a transgene using synthetic 5′UTRs.

The present invention is directed to methods, compositions and systems for capturing and analyzing sequence information contained in targeted regions of a genome. Such targeted regions may include exomes, partial exomes, introns, combinations of exonic and intronic regions, genes, panels of genes, and any other subsets of a whole genome that may be of interest.

A method for the ligation of expressed proteins which utilizes inteins, for example the RIR1 intein from Methanobacterium thermotrophicum, is provided. Constructs of the Mth RIR1 intein in which either the C-terminal asparagine or N-terminal cysteine of the intein are replaced with alanine enable the facile isolation of a protein with a specified N-terminal, for example, cysteine for use in the fusion of two or more expressed proteins. The method involves the steps of generating a C-terminal thioester-tagged target protein and a second target protein having a specified N-terminal via inteins, such as the modified Mth RIR1 intein, and ligating these proteins. A similar method for producing a cyclic or polymerized protein is provided. Modified inteins engineered to cleave at their C-terminus or N-terminus, respectively, and DNA and plasmids encoding these modified inteins are also provided.

This invention generally relates to a method for developing, generating and selecting human embryonic stem (hES)-like pluripotent cells using transgenic expression of intronic microRNA-like RNA agents. More particularly, the present invention relates to a method and composition for generating a non-naturally occurring intron and its intronic components capable of being processed into mir-302-like RNA molecules in mammalian cells and thus inducing certain specific gene silencing effects on differentiation-related and fate-determinant genes of the cells, resulting in reprogramming the cells into a pluripotent embryonic stem (ES)-cell-like state. The ES-like cells so obtained are strongly express hES cell markers, such as Oct3/4, SSEA-3 and SSEA-4, and can be guided into various tissue cell types by treating certain hormones and/or growth factors under a feeder-free cell culture condition in vitro, which may be used for transplantation and gene therapies. Therefore, the present invention offers a simple, effective and safe gene manipulation approach for not only reprogramming somatic cells into ES-like pluripotent cells but also facilitating the maintenance of pluripotent and renewal properties of ES cells under a feeder-free cell culture condition, preventing the tedious retroviral insertion of four large transcription factor genes into one single cell as used in the previous iPS methods.

The invention relates to methods for the identification and use of introns with gene expression enhancing properties. The teaching of this invention enables the identification of introns causing intron-mediated enhancement (IME) of gene expression. The invention furthermore relates to recombinant expression construct and vectors comprising said IME-introns operably linked with a promoter sequence and a nucleic acid sequence. The present invention also relates to transgenic plants and plant cells transformed with these recombinant expression constructs or vectors, to cultures, parts or propagation material derived there from, and to the use of same for the preparation of foodstuffs, animal feeds, seed, pharmaceuticals or fine chemicals, to improve plant biomass, yield, or provide desirable phenotypes.

Provided are tricyclo-DNA (tc-DNA) AON and methods employing tc-DNA AON for modifying splicing events that occur during pre-mRNA processing. Tricyclo-DNA (tc-DNA) AON are described that may be used to facilitate exon skipping or to mask intronic silencer sequences and/or terminal stem-loop sequences during pre-mRNA processing and to target RNase-mediated destruction of processed mRNA. Tc-DNA AON described herein may be used in methods for the treatment of Duchenne Muscular Dystrophy by skipping a mutated exon 23 or exon 51 within a dystrophin gene to restore functionality of a dystrophin protein; in methods for the treatment of Spinal Muscular Atrophy by masking an intronic silencing sequence and/or a terminal stem-loop sequence within an SMN2 gene to yield modified functional SMN2 protein, including an amino acid sequence encoded by exon 7, which is capable of at least partially complementing a non-functional SMN1 protein; and in methods for the treatment of Steinert's Myotonic Dystrophy by targeting the destruction of a mutated DM1 mRNA comprising 3′-terminal CUG repeats.

Elucidating the function of proteins in mammalian cells is particularly challenging due to the inherent complexity of these systems. Methods to study protein function in living cells ideally perturb the activity of only the protein of interest but otherwise maintain the natural state of the host cell or organism. Ligand-dependent inteins offer single-protein specificity and other desirable features as an approach to control protein function in cells post-translationally. Some aspects of this invention provide second-generation ligand-dependent inteins that splice to substantially higher yields and with faster kinetics in the presence of the cell-permeable small molecule 4-HT, especially at 37° C., while exhibiting comparable or improved low levels of background splicing in the absence of 4-HT, as compared to the parental inteins. These improvements were observed in four protein contexts tested in mammalian cells at 37° C., as well as in yeast cells assayed at 30° C. or 37° C. The newly evolved inteins described herein are therefore promising tools as conditional modulators of protein structure and function in yeast and mammalian cells.

Disclosed are useful constructs and methods for the expression of proteins using primary translation products that are processed within a recombinant host cell. Constructs comprising a single open reading frame (sORF) are described for protein expression including expression of multiple polypeptides. A primary translation product (a pro-protein or a polyprotein) contains polypeptides such as inteins or hedgehog family auto-processing domains, or variants thereof, inserted in frame between multiple protein subunits of interest. The primary product can also contain cleavage sequences such as other proteolytic cleavage or protease recognition sites, or signal peptides which contain recognition sequences for signal peptidases, separating at least two of the multiple protein subunits. The sequences of the inserted auto-processing polypeptides or cleavage sites can be manipulated to enhance the efficiency of expression of the separate multiple protein subunits. Also disclosed are independent aspects of conducting efficient expression, secretion, and/or multimeric assembly of proteins such as immunoglobulins. Where the polyprotein contains immunoglobulin heavy and light chain segments or fragments capable of antigen recognition, in an embodiment a selectable stoichiometric ratio is at least two copies of a light chain segment per heavy chain segment, with the result that the production of properly folded and assembled functional antibody is made. Modified signal peptides, including such from immunoglobulin light chains, are described.

The invention provides an overexpression porcine co-stimulatory 4-1BB vector and application thereof. PCR (polymerase chain reaction) amplification is performed on a left homologous arm and a right homologous arm of an intron 1 of a rosa26 gene, a 4-1BB regulatory sequence and an OCT4 specific promoter; the left homologous arm, a 4-1BB expression cassette, LoxP locus-contained Cre and Neo expression cassettes, the right homologous arm and negative selection DTA diphtheria toxin are connected in sequence to obtain a 4-1BB homologous recombinant vector p4BOCNDR; the vector and a CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated) targeting vector of sgRNA (small guide ribonucleic acid) containing the intron 1 of the specific targeting porcine rosa26 gene are transferred together into a porcine fetus fibroblast; by taking a positive cell as a donor cell and an oocyte as a recipient cell, a cloned embryo is obtained through a somatic cell nuclear transfer technique; the cloned embryo is transplanted into a porcine uterus for fetation to obtain a transgenic pig integrating a 4-1BB gene at the fixed point of a first intron of the rosa26 gene and automatically deleting a marker gene.

A method and compound for suppressing an immune response in a mammalian subject, for the treatment or prevention of an autoimmune condition or transplantation rejection are disclosed. The compound is an antisense oligonucleotide analog compound having a targeting sequence complementary to a preprocessed CTLA-4 mRNA region identified by SEQ ID NO: 22 in SEQ ID NO: 1, spanning the splice junction between intron 1 and exon 2 of the preprocessed mRNA of the subject. The compound is effective, when administered to a subject, to form within host cells, a heteroduplex structure (i) composed of the preprocessed CTLA-4 mRNA and the oligonucleotide compound, (ii) characterized by a Tm of dissociation of at least 45° C., and (iii) resulting in an increased ratio of processed mRNA encoding ligand-independent CTLA-4 to processed mRNA encoding full-length CTLA-4.

A self-cleaving element for use in bioseparations has been derived from a naturally occurring, 43 kDa protein splicing element (intein) through a combination of protein engineering and random mutagenesis. A mini-intein (18 kDa) previously engineered for reduced size had compromised activity and was therefore subjected to random mutagenesis and genetic selection. In one selection a mini-intein was isolated with restored splicing activity, while in another, a mutant was isolated with enhanced, pH-sensitive C-terminal cleavage activity. The enhanced cleavage mutant has utility in affinity fusion-based protein purification. The enhanced splicing mutant has utility in purification of proteins such as toxic proteins, for example, by inactivation with the intein in a specific region and controllable splicing. These mutants also provide new insights into the structural and functional roles of some conserved residues in protein splicing. Thus, disclosed and claimed are: a genetic system and self-cleaving inteins therefrom; bioseparations employing same; protein purification by inactivation with inteins in specific regions and controllable intein splicing; methods for determining critical, generalizable residues for varying intein activity; and products.

The invention discloses a method for pig CMAH gene specific knockout through CRISPR-Cas9 and sgRNA for specially targeting a CMAH gene. A target sequence of the sgRNA for specially targeting the CMAH gene on the CMAH gene meets a 5'-N(20)NGG-3' sequence arrangement rule, wherein the N(20) represents 20 continuous bases, and each N represents A, T, C or G; the target sequence on the CMAH gene locates at a 5-exon coding area or a junction position of adjacent introns of an N end of the CMAH gene; and the target sequence on the CMAH gene is unique. The sgRNA is used in a method for pig CMAH gene specific knockout through the CRISPR-Cas9, the pig CMAH gene can be rapidly, accurately, efficiently and specifically knocked out, and the problems of the long period and high cost of construction of a CMAH gene knockout pig are effectively solved.

The present invention discloses a pig SLA-1 gene knockout method using CRISPR-Cas9 specificity and sgRNA used for specific targeting SLA-1 gene. The target sequence of the sgRNA used for specific targeting SLA-1 gene in the SLA-1 gene complies with a 5'-N (20) NGG-3' sequence arrangement rule, wherein N (20) represents 20 consecutive basic groups, wherein each N represents a A or T or C or G; the target sequence in the SLA-1 gene is located at the four exon coding regions of the N-terminal of the SLA-1 gene or the junction of adjacent introns; and the target sequence in the SLA-1 gene is unique. The sgRNA used in the pig SLA-1 gene knockout method using CRISPR-Cas9 specificity, may fast, accurately, efficiently, and specifically knockout pig SLA-1 gene, effectively solve long cycle and high cost in construction of SLA-1 gene knockout pig.

By this invention, novel nucleic acid sequences are provided, wherein said nucleic acid sequence is a genomic sequence of a plant desaturase encoding sequence. Also provided in the present invention are the promoter and intron sequences of the desaturase genomic sequences. Furthermore, recombinant DNA constructs employing the polynucleotide sequences are provided. The instant invention also provides methods for the modification of fatty acid compositions in host plant cells.

The invention discloses a method for CRISPR-Cas9 specific knockout of a pig GGTA1 gene and sgRNA for a specific targeted GGTA1 gene. The target sequence of the sgRNA for the specific targeted GGTA1 gene on the GGTA1 gene is in accordance with the sequence arrangement rule of 5'-N(20)NGG-3', wherein N(20) represents 20 continuous bases, and each N represents A or T ot C or G; the target sequence on the GGTA1 gene is positioned at a junction of 5 exon coding areas and/or adjacent introns of the N-end of the GGTA1 gene; and the target sequence on the GGTA1 gene is unique. According to the method for CRISPR-Cas9 specific knockout of the pig GGTA1 gene via sgRNA, specific knockout of the pig GGTA1 gene can be rapidly and accurately realized with high efficiency, and the problems of long period and high cost for constructing the knockout of the pig GGTA1 gene can be effectively solved.

The invention discloses a method for specifically removing a pig SALL1 gene by CRISPR-Cas9 and sgRNA used for the specific targeting SALL1 gene. The sgRNA of the specific targeting SALL1 gene has a target sequence on the SALL1 gene which accords with a sequence arrangement rule of 5'-N(20)NGG-3', wherein N(20) represents 20 continuous basic groups, each N represents A or T or C or G. The target sequence on the SALL1 gene is positioned in the three exon code areas at the N end of the SALL1 gene or at the junction of the exons and the adjacent intrones. The target sequence on the SALL1 gene is unique. The sgRNA is used in the method for specifically removing the pig SALL1 gene by the CRISPR-Cas9, the pig SALL1 gene can be removed, and the problems if long period and high cost for SALL1 gene removal are effectively solved.

The invention discloses a method using CRISPR-Cas9 to specifically knock off pig PDX1 gene and sgRNA of PDX1 gene for specific targeting. The target sequence of sgRNA of PDX1 gene for specific targeting on PDX1 gene is accord with the sequence alignment rule of 5'-N(20)NGG-3', wherein N(20) represents 20 continuous bases, and each N represent A, T, C or G; the target sequence of PDX1 gene is arranged in the first exon coding region on the N terminal of PDX1 gene or a junction between the coding region and neighbored intron, and the target sequence of PDX1 gene is unique. The provided method can rapidly, precisely, efficiently and specifically knock off PDX1 gene of pigs, and solves the problems of long period and high cost of pig PDX1 gene knocking.

The invention provides a genome editing method based on a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) system and belongs to the technical field of gene editing. The genome editing method provided by the invention utilizes introns to express guide RNA molecules in the CRISPR genome editing system and adopts a principle that one or more than one tRNA-gRNA or crRNA series unitsare arranged in the introns of an encoded gene, and after the introns form a fusion gene with Cas9 or Cpf1, a promoter can be used for driving. The genome editing method provided by the invention hasthe benefits that a cellular endogenous mRNA splicing system and a cellular endogenous tRNA processing system are skillfully utilized, and other elements are not required to be added, so that not onlyis the safety higher, but also more simplicity can be realized; the synchronous expression of a plurality of guide RNAs with the Cas9 or the Cpf1 is realized by utilizing the one promoter, so that anexisting CRISPR editing system is simplified, moreover, the efficiency and the ability of simultaneously editing multiple target points of the CRISPR editing system are improved.