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Methods of producing a library and methods of selecting polynucleotides of interest

a polynucleotide and library technology, applied in the field of producing a library and selecting polynucleotides of interest, can solve the problems of cloning many disease genes with negative or toxic effects on cell proliferation, revertant lines are typically difficult to identify and separate from the majority of rapidly growing transformed parental cells, and each of these classical genetic approaches is limited.

Inactive Publication Date: 2006-07-20
UNIVERSITY OF ROCHESTER
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Benefits of technology

The present invention provides methods for high efficiency cloning and producing a library using a linear DNA virus vector such as vaccinia virus vector. These methods can be used to clone polynucleotides that negatively affect cell viability, select polynucleotides that encode epitopes, or modify the phenotype of a cell. The invention also provides kits for producing an antisense expression library and a protein expression library. These methods and kits can be useful in research and development of new therapies and treatments for diseases.

Problems solved by technology

Each of these classical genetic approaches is limited in the type of gene which can be isolated or in the extensive time and labor required.
However, a major technical limitation to the cloning of many disease genes is their negative or toxic effect on cell proliferation when present in multiple copies, such as when carried on a vector.
However, revertant lines typically are difficult to identify and separate from the majority of rapidly growing transformed parental cells.
In addition, the method may preclude the isolation of certain classes of revertants.
The selection procedure may itself induce epigenetic or cytogenetic changes, thus further complicating the identification of genes responsible for the revertant phenotype.
However, the approach is limited to particular transformation mechanisms because the prolonged dye retention phenotype is neither essential nor sufficient for cell transformation.
None of these approaches, however, offers a way to directly assess gene function as a method of identifying genes of interest, especially negative regulators of proliferation.
The identification of toxic molecules such as tumor suppressor genes and other inhibitors of cell proliferation to screen for potential new drugs is difficult using current technology.
Those negative or toxic mutations that result in inhibition of cell growth or in cell death may be masked in a library or other population of cells due to the low efficiency of transfection.
Additionally, such negative or toxic mutations cannot be selected for or screened using current technology because cells expressing such variants are lost from the population of transformants.
However, this approach is labor-intensive, is not applicable to certain situations, and has met with varied success depending on the cell type and origin of the promoter utilized.
As alluded to above, there are methods to identify positive regulators of cell growth such as oncogenes, but approaches to isolate toxic genes such as tumor suppressor genes are limited.
This method does not distinguish recipients expressing a gene or cDNA of interest, e.g., a negative or toxic variant, from the remaining recipients.
However, the mouse has not been used for large scale classical genetic mutational analysis because random mutational screening and analysis is very cumbersome and expensive due to long generation times and maintenance costs.
A disadvantage in using animal models for the identification of genes is the need to establish a transgenic animal line for each mutational event.
This disadvantage is alleviated in part by using embryonic stem (ES) cell lines because mutational events may be screened in vitro prior to generating an animal.
Such methods, however, have major drawbacks when the object is to clone mRNAs of relatively low abundance from cDNA libraries.
For example, using direct in situ colony hybridization, it is very difficult to detect clones containing cDNA complementary to mRNA species present in the initial library population at less than one part in 200.
There appear to be two principal reasons for this: First, the existing technology (Okayama, H. et al., Mol. Cell. Biol. 2:161-170 (1982)) for construction of large plasmid libraries is difficult to master, and library size rarely approaches that accessible by phage cloning techniques.
Second, the existing vectors are, with one exception (Wong, G. G., et al., Science 228:810-815 (1985)), poorly adapted for high level expression, particularly in COS cells.
These methods often are inefficient and tedious and require multiple rounds of screening to identify full-length or overlapping clones.
Prior screening methods based upon expression of fusion proteins are inefficient and require large quantities of monoclonal antibodies.
Such drawbacks are compounded by use of inefficient expression vectors, which result in protein expression levels that are inadequate to enable efficient selection.
However, this method is limited to the isolation and cloning of proteins which are expressed and transported to the cell surface, whose expression does not adversely affect cell viability, and for which specific antibody has been isolated.
Although homologous recombination is efficient for transferring previously isolated foreign DNA of relatively small size into vaccinia virus, the method is much less efficient for transferring large inserts, for constructing libraries, and for transferring foreign DNA which is deleterious to bacteria.
The cloning methods and the selection methods above have a number of drawbacks and limitations.

Method used

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  • Methods of producing a library and methods of selecting polynucleotides of interest
  • Methods of producing a library and methods of selecting polynucleotides of interest
  • Methods of producing a library and methods of selecting polynucleotides of interest

Examples

Experimental program
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example 1

Construction and Characterization of Vaccinia Expression Vectors

[0579] This example describes the construction and characterization of a new set of direct ligation vectors designed to be universally applicable for the generation of chimeric vaccinia genomes. The aim was to modify the genome of vNotI / tk so as to acquire direct ligation vectors which are more universally useful. First, the insertion site was changed by placing the sites for two unique restriction enzymes at the beginning of the thymidine kinase gene. This allows one to fix the orientation of the insert polynucleotide (e.g. DNA) and eliminates the production of contaminating wild type genomes after religation of viral arms. Second, in order to generate a direct ligation vector which would express high levels of protein, the thymidine kinase gene was preceded by a strong constitutive vaccinia virus promoter.

[0580] These new ligation vectors contain a pair of unique restriction sites, NotI and ApaI, to eliminate religa...

example 2

Trimolecular Recombination

[0618] Production of an Expression Library. This example describes a tri-molecular recombination method employing modified vaccinia virus vectors and related transfer plasmids that generates close to 100% recombinant vaccinia virus and, for the first time, allows efficient construction of a representative DNA library in vaccinia virus.

[0619] Construction of the Vectors. The previously described vaccinia virus transfer plasmid pJ / K, a pUC 13 derived plasmid with a vaccinia virus thymidine kinase gene containing an in-frame Not I site (Merchlinsky, M. et al., Virology 190:522-526), was further modified to incorporate a strong vaccinia virus promoter followed by Not I and Apa I restriction sites. Two different vectors, p7.5 / tk and pEL / tk, included, respectively, either the 7.5K vaccinia virus promoter or a strong synthetic early / late (E / L) promoter (FIG. 1). The Apa I site was preceded by a strong translational initiation sequence including the ATG codon. Th...

example 3

Direct Selection Using Target Epitope-Specific Cytotoxic T Cells

[0641] In this example, a model system was assayed to determine the level of enrichment that can be obtained through a procedure that selects for DNA recombinants that encode the target epitopes of tumor specific cytotoxic T cells.

Methods and Results

[0642] A specific vaccinia recombinant that encodes a well characterized ovalbumin peptide (SIINFEKL) (SEQ ID NO:26) was diluted with non-recombinant virus so that it constituted either 0.2%, 0.01%, or 0.001% of viral pfu. This ovalbumin peptide is known to be processed and presented to specific CTL in association with the murine class I MHC molecule H-2Kb. An adherent monolayer of MC57G cells that express H-2Kb were infected with this viral mix at m.o.i.=1 (approximately 5×105 cell / well). MC57G cells do not themselves express ovalbumin peptide, but do express H-2Kb, which allows them to associate with and present ovalbumin peptide to the T cells.

[0643] Following 12 hou...

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Abstract

The present invention relates to a high efficiency method of introducing DNA into linear DNA viruses such as poxvirus, a method of producing libraries in linear DNA viruses such as poxvirus, and methods of selecting polynucleotides of interest based on cell nonviability or other phenotypes.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application is a divisional of U.S. application Ser. No. 09 / 818,991, filed Mar. 28, 2001, which claims the benefit of U.S. Provisional Appl. No. 60 / 192,586, filed Mar. 28, 2000; U.S. Provisional Appl. No. 60 / 203,343, filed May 10, 2000; U.S. Provisional Appl. No. 60 / 263,226, filed Jan. 23, 2001; and U.S. Provisional Appl. No. 60 / 271,426, filed Feb. 27, 2001; each disclosure of which is herein incorporated by reference in its entirety.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention relates to a high efficiency method of introducing DNA into poxvirus, a method of producing libraries in poxvirus, and methods of isolating polynucleotides (of interest based on cell nonviability or screening methods. [0004] 2. Background Art [0005] Identification of Disease Genes. In the past decade it has become apparent that many diseases result from genetic alterations in signaling pathways. These inclu...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C40B40/08C12N15/09G01N33/53C12N15/10C12N15/34C12N15/39C12Q1/02C12Q1/68G01N37/00
CPCC12N15/1034C12N15/1079C12N2799/023
Inventor ZAUDERER, MAURICE
Owner UNIVERSITY OF ROCHESTER
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