Leland Stanford Junior University

Children's Medical Center Corporation

Liqun Luo

Zhigang He

Jing Wang

Eric Hoopfer

Ryan Watts

Hui Zong

Qiwei Zhai

Ruby Yanru Tsai

Bosiljka Tasic

Simon Hippenmeyer

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.

Site-directed integration of transgenes in mammals

Recombination in mammalian somatic cell chromosomes is promoted and marked by a method called mosaic analysis with double marker (MADM). Mouse 'knock-in' techniques are used to create pairs of chromosomes in which recombinase target sites are placed at homologous chromosomal locations. The knock-in constructs are engineered so that cellular markers, such as green or red fluorescent protein (GFP or RFP), are only expressed after recombinase-induced recombination. This system provides high-sensitivity detection of recombinase-induced mitotic recombination, even down to the single cell level. When this recombination is induced in a mouse heterozygous for a mutation in a gene distal to the 'knock-in' locus on the same chromosome, it results in homozygosity of this mutation in the labeled cells. This allows the analysis in singly-labeled neurons of genes whose pleiotropic effects might otherwise result in early lethality.

Somatic recombination

Reduced degeneration of an axon predetermined to be subject to degenerative neuropathy in a term patient is effected by contacting the axon in situ with an effective amount of a ubiquitin-proteasome system (UPS) inhibitor sufficient to reduce degeneration of the axon; and detecting a resultant reduction in the degeneration of the axon in situ.

Reducing axon degeneration with proteasome inhibitors

Growing community of inventors

Stanford, CA, United States of America

Liqun Luo