Methods of preforming homologous recombination based modification of nucleic acids in recombination deficient cells and use of the modified nucleic acid products thereof

Abstract

A simple method for modifying genes in a recombination deficient host cell is disclosed. Such modifications include generating insertions, deletions, substitutions, and / or point mutations at any chosen site in the independent origin based cloning vector. The modified gene is contained in an independent origin based cloning vector that is used to introduce a modified heterologous gene into a cell. Such a modified vector may be used in the production of a germline transmitted transgenic animal, or in gene targeting protocols in eukaryotic cells. In particular, high throughput methodology is provided for generating the modified the independent origin based cloning vectors of the present invention.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application is a Continuation-In-Part of copending U.S. Ser. No. 09 / 356,987 filed Jul. 20, 1999 which is a Continuation-In-Part of copending U.S. Ser. No. 09 / 102,490 filed Jun. 22, 1998 which is a non-provisional application claiming the priority of provisional U.S. Ser. No. 60 / 050,535 filed Jun. 23, 1997, the disclosures of which are hereby incorporated by reference in their entireties. Applicants claim the benefits of these applications under 35 U.S.C. §§119(e) and 120.GOVERNMENTAL SUPPORT [0002] The research leading to the present invention was supported, at least in part, by a grant from the National Science Foundation Grant No. MCB-9316625, by NINDS PHS 30532, and NIH MSTP grant GM07739. Accordingly, the Government may have certain rights in the invention.FIELD OF THE INVENTION [0003] This invention relates generally to methods of modifying genes with specificity in recombination deficient cells by transiently enabling ...

AI Technical Summary

Benefits of technology

[0034] The present invention further provides methods of selectively modifying a particular nucleotide sequence of an independent origin based cloning vector (IOBCV) that is contained in a recombination deficient host cell that are particularly conducive for high throughput procedures. These high throughput procedures are preferentially performed almost entirely in liquid rather than on plates thereby facilitating the modification of multiple BACs at one time, (e.g., performing separate modifications to different BACs at the same time).

[0036] The conditional replication shuttle vector contains a nucleic acid that selectively integrates into the particular nucleotide sequence when the recombination protein is expressed, thereby forming a co-integrate. The nucleic acid that selectively integrates into the particular nucleotide sequence and the nucleic acid encoding the recombination protein are positioned on the conditional replication shuttle vector such that upon resolution of the co-integrate, the nucleic acid encoding the recombination protein remains with the conditional replication shuttle vector. Thus, growing the host cell under conditions in which the conditional replication shuttle vector cannot replicate dilutes out the conditional replication shuttle vector encoding the recombination protein, and thereby prevents further (undesirable) recombination events in the recombination deficient cells to occur.

[0052] Accordingly, it is a principal object of the present invention to provide a method for readily and specifically modifying an independent origin based cloning vector in a recombination deficient host cell.

[0064] It is still a further object of the present invention to provide a method for readily and specifically modifying an independent origin based cloning vector in a recombination deficient host cell under conditions that allow multiple modifications of IOBCVs at the same time.

Problems solved by technology

On the other hand, the use of such large genomic transgenes has several practical problems.

For example, the size of the transgene is presently limited due to constraints on the sequence length that can be cloned and stably maintained in a conventional plasmid or a cosmid.

Thus DNA sequences suspected of being nonessential are often omitted when designing the constructs to be transferred because of the size limitation.

However, there are several critical limitations in the YAC system including difficulties in manipulating YAC DNA, chimerism and clonal instability [Green et al., Genomics, 11:658 (1991); Kouprina et al., Genomics 21:7 (1994); Larionov et al., Nature Genet.

As a result, generating transgenic mice with an intact YAC remains a challenging task [Burke et al., Science 236:806; Peterson et al., Trends Genet.

However, while PACs and BACs have cloning capacities up to 350 kb, performing homologous recombination to introduce mutations into a gene of interest has not been demonstrated [Peterson et al., TIG 13:61-66].

Indeed, although BACs or PACs have become an important source of large genomic DNA in genome research, there are still no methods available to modify the BACs or PACs.

Furthermore, no germline transmission of intact BACs or PACs in transgenic mice have been reported.

These, as well as other disadvantages of BACs and PACs greatly limit their potential use for functional studies.

However, in the vertebrate system, the rate of homologous recombination is very low, as compared to random integration.

However, the rate of homologous recombination for some gene loci in ES cells is still extremely low (<1%), the procedure is labor intensive, and the cost of generating targeted mutant mice is very expensive.

Moreover, since there are no ES cells available for vertebrates other than mice, gene targeting in a germline is still not possible for other vertebrates.

A major limitation for gene transfer procedures in vertebrate cells such as gene targeting is the low targeting frequency.

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