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

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...

AI Technical Summary

Benefits of technology

[0041] In accordance with one aspect of the present invention, there is provided a method of high efficiency cloning using a linear DNA virus vector such as vaccinia virus vector, comprising tri-molecular recombination.

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.

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