Inducible, reversible gene knockdown
The Transgenic Mouse Facility (TMF) has acquired a specially engineered mouse embryonic stem cell line (the KH2 line) that is designed for efficient insertion of small RNA coding sequences (e.g., shRNA) into a specific locus in the mouse genome and to allow expression of such sequences in an inducible and reversible fashion.
In collaboration with Cancer Center members Anand Ganesan and Bogi Andersen, the TMF recently completed a proof-of-principle experiment with these cells. Expression of the Apc gene was knocked down by inserting an shRNA and inducing its expression with doxycycline added to the culture media.
Targeted cells were injected into mouse blastocysts to make chimeric mice. These mice were treated with doxycycline in their drinking water, which resulted in an easily detectable hair overgrowth phenotype.
Sperm cryopreservation and in vitro fertilization
The TMF has successfully cryopreserved more than 80 mouse strains for UC Irvine investigators over the last 2 years, after importing new techniques for sperm freezing and in vitro fertilization developed by the Jackson Laboratory and the Center for Animal Resources and Development at Kumamoto University, Japan.
TMF manuscript accepted for publication
In collaboration with the laboratories of cancer center associate member Grant MacGregor and former member Doug Wallace, the TMF conducted a study to compare the efficiencies of two C57BL/6 substrains when used as the source of blastocysts for the injection of ES cells to make chimeric mice.
Our manuscript, titled “Comparison of male chimeric mice generated from microinjection of JM8.N4 embrynic stem cells into C57BL/6J and C57BL/6NTac blastocysts,” has been accepted for publication in Transgenic Research.
Bringing recombineering to UC Irvine
The TMF is spearheading an effort to establish a new core facility at UC Irvine that will use the flexible and efficient DNA cloning technique known as recombineering to produce targeting vectors and other constructs.
We have conducted an on-line survey to gauge demand and intend to present a business proposal to the Office of Research in the near future.