Site-directed integration of transgenes in mammals

摘要

The present disclosure provides a method of making a mammal (e.g., a rodent, such as a mouse) by integrating an intact polynucleotide sequence into a specific genomic locus of the mammal to result in a transgenic mammal. A transgenic mammal made by the methods of the present disclosure would contain a known copy number (e.g., one) of the inserted polynucleotide sequence at a predetermined location. The method involves introducing a site-specific recombinase and a targeting construct, containing a first recombination site and the polynucleotide sequence of interest, into the mammalian cell. The genome of the cell contains a second recombination site and recombination between the first and second recombination sites is facilitated by the site-specific, uni-directional recombinase. The result of the recombination is site-specific integration of the polynucleotide sequence of interest in the genome of the mammal. This inserted sequence is then also transmitted to the progeny of the mammal.

主权项

1. A method of inserting a polynucleotide sequence into a genome of a murine cell, comprising:
introducing a circular nucleic acid into said murine cell,
wherein said circular nucleic acid comprises said polynucleotide sequence and a first unidirectional recombination site, andwherein said genome of said murine cell comprises a second unidirectional recombination site at the intergenic Hipp11 (H11) locus on mouse chromosome 11; introducing an mRNA a nucleic acid encoding a site-specific, unidirectional recombinase into said cell; and maintaining said cell under conditions that facilitate recombination between said first and second unidirectional recombination sites mediated by said unidirectional recombinase, wherein said method results in a site-specific integration of said polynucleotide sequence into the genome of said murine cell.