Cellular and Integrative Physiology
Reahum Paik, B.S
Mouse Genome Engineering & Transgenic Facility
Viability, timeline, suggestions, plan overview, and risks associated with any transgenic mouse project will be analyzed and discussed with the investigators.
The facility is well equipped and resourced to clone any DNA fragment in any type of vectors. Molecular cloning projects are not limited to transgenics, and can include AAV or Lentivirus plasmid vectors, siRNA vectors, Drosophila targeting vectors, or any shuttle plasmids. The price will vary depending on the size, type/number of inserts, and any associated difficulties. The facility will also help in synthesizing large ODN or dsDNA using gBlock or other available technologies from commercial sources.
Pronuclear injections target 250 to 300 zygotes. BAC DNA or any other plasmid DNA can be injected to generate transgenic mouse lines. For CRISPR projects, we use crRNA with tracrRNA and Cas9 protein. We recommend IDT alt-R production of your crRNA. CRISPR service requests can be submitted partially or entirely, depending on PI’s resources and/or preferences. If only injection is requested, the facility will require that the PIs show T7E1 assay picture for crRNAs containing amplicon from transfected and drug selection tested mammalian cell genomic DNA. crRNA of the plasmid needs to be sequenced. The facility will also perform toxicity test on few injected zygote cultures before the actual injection, and individual blastocyst sequencing will be requested. Alternatively, all the validation tests can be performed by the facility at a reasonable cost.
Cryopreservation of mouse sperm service is offered by the facility. Sperms from up to 3 male mice are collected and stored in LN2. Partial IVF is recommended to verify the sperm viability, but not obligatory.
PCR based mouse tissue genotyping service is currently available at the facility at reasonable costs.
Cryopreserved Sperm & Embryo Resuscitation
The facility can resuscitate frozen sperms by incubating with donor eggs via IVF, then transferring the fertilized eggs into a foster mother. Frozen embryos can be also resuscitated by transferring into foster mother’s oviduct or uterus depending on the developmental stage of the cryopreserved embryos.
In Vitro Fertilization
Mouse eggs can be fertilized with freshly collected sperms via IVF, then transferred to foster mothers.
Manzoor Bhat, M.S., Ph.D.
Professor and Chair, Cellular & Integrative Physiology
Teppei Fujikawa, Ph.D.