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

a technology of recombination deficiency cells and homologous recombination, which is applied in the direction of peptides, applications, peptide sources, etc., can solve the problems of critical limitations in the yac system, the size of the transgene is presently limited, and the use of such large genomic transgenes has several practical problems

Inactive Publication Date: 2005-09-22
HEINTZ NATHANIEL +3
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  • Abstract
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Benefits of technology

[0064] It is still a further object of the present invention to provide a method for readily and specifically modifying an independent origi

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 [Peter

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

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example 1

Homologous Recombination Based Modification in E. coli and Germline Transmission in Transgenic Mice of an 131 Kilobase Bacterial Artificial Chromosome

Introduction

[0209] Bacterial based artificial chromosomes, such as Bacterial artificial chromosomes (BACs) and P-1 derived artificial chromosomes (PACs), are circular bacterial plasmids that may propogate as large as 300 kb of exogenous genomic DNA [Shizuya et al., PNAS, 89:8794-8797, (1992); Ioannou et al., Nature Genet., 6:84-90 (1994)]. For the majority of BAC and PAC libraries, the average size of the insert is 130-150 kb. There are several advantages of using bacterial based artificial chromosomes for genomic and functional studies, compared to the yeast based system (i.e. YACs): First, BAC and PAC libraries are much easier to construct due to higher cloning efficiency. Second, BACs and PACs ate propagated in recombination deficient E. coli host cells, so they have high stability and minimal chimerism. No rearrangements have bee...

example 2

BAC Mediated Gene Dosage Analysis: A Role for Zipro1 (RU49 / Zfp38) in Progenitor Cell Proliferation in Cerebellum and Skin

Introduction

[0331] Analysis of loss-of-function phenotypes has played a central role in the discovery of complex morphogenetic pathways in a variety of organisms. For example, the seminal loss-of-function screens for genes affecting cell cycle traverse in yeast [Hartwell et al, Science 183(120):46-51 (1974)] and for mutations affecting early D. melanogaster development [Nusslein-Volhard and Wieschaus, Nature 287:795-801 (1980)] have provided the basis for our current understanding of cell division and of embryonic patterning. However, in both cases, it was readily appreciated that loss-of-function genetics would not yield all the genes in the pathway under study, and alternative strategies for genetic analysis were therefore devised. Thus, high copy number suppression screens have been highly successful in identifying additional genes important for cell division...

example 3

Rapid Modification and High Throughput Resolution of Bacterial Artificial Chromosomes in Liquid

Introduction

[0364] Traditionally, overexpression of cDNA in eukaryotic cells and transgenic mice has been widely used for the study of gene function and regulation. However, the cDNA itself is often missing important elements for regulation of gene activity, such as high-level, tissue-specific, and integration site-independent expression of the transgene. Those elements such as enhancers, locus control regions (LCR), and insulators, may reside at a large distance (>50 kb) from the gene itself. A intact genomic loci as a transgene will be essential for this expression. Bacterial artificial chromosome (BACs) and P-1 derived artificial chromosomes (PACs) have become a widespread and powerful resource in manipulating the large genomic DNA in E. coli. However, although BAC transgenic technology has been used for studying gene function and regulation, the efficiency for modifying and resolving...

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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 ...

Claims

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Application Information

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IPC IPC(8): A01K67/027C07K14/47C12N15/09C12N15/10C12N15/64C12N15/65C12N15/70C12N15/85C12N15/86C12N15/90
CPCA01K67/0275C12N2840/206A01K2217/05A01K2217/075A01K2227/105A01K2267/0356C07K14/4705C12N15/102C12N15/64C12N15/65C12N15/70C12N15/8509C12N15/902C12N2800/204C12N2800/30C12N2840/203A01K67/0276
Inventor HEINTZ, NATHANIELMODEL, PETERYANG, XIANGDONGGONG, SHIAOCHING
Owner HEINTZ NATHANIEL
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