This proposal develops powerful new methods for obtaining efficient site-specific integration at pre-determined genomic sequences. The work elaborates on a successful line of research in my laboratory involving recombination mediated by phage integrases at their compact (30 - 40-bp) attB and attP recognition sites. We have demonstrated that this reaction represents the most efficient site-specific integration system developed to date for higher cells. In the proposed strategy, integration takes place between an attB site present on an incoming DNA vector and a naturally-occurring attP-like sequence located in the mouse genome. The result is covalent integration of the incoming DNA into a pre-specified position in the mouse genome. Directed evolution of phage integrases is used to produce a large library of integration tools of unprecedented efficiency and specificity. With this system, a custom integration tool can be designed for any genomic sequence and used to integrate a DNA insert of large or small size at high efficiency. The DNA to be inserted can be custom designed as desired. Because the method is site-specific, the location of the insertion is automatically known. We develop the appropriate DNA shuffling and genetic screening methods here for generation of a library of custom integration tools. The strategy is scalable to reach essentially all genes in the mouse genome.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21HG002571-02
Application #
6526816
Study Section
Special Emphasis Panel (ZRG1-MGN (01))
Program Officer
Graham, Bettie
Project Start
2001-09-30
Project End
2004-08-31
Budget Start
2002-09-01
Budget End
2003-08-31
Support Year
2
Fiscal Year
2002
Total Cost
$153,580
Indirect Cost
Name
Stanford University
Department
Genetics
Type
Schools of Medicine
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305