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We has been using the CRISPR technology to create genome-edited mice since 2013.  We have made mutant mice with point mutation, exon deletion, epitope tag insertion and insertions of loxP, docking site, EGFP and cre at endogenous loci or transgenes at Rosa26.  Our fees for CRISPR-based germline modifications are very competitive.

We can help you with CRISPR project design, screening strategy and result analysis. We need to know from you the gene of interest and type of mutation to get started with the design which is free.

We have been producing transgenic mice since 1992. We can design your transgenic project for random integration in the mouse genome. The investigator should linearize the transgene away from the vector and gel-purify the construct, determine the concentration accurately and resuspend the transgene at 1 ug/ul in 20 ul TE buffer.

Transgenes should include promoter, enhancer, gene to be expressed, splice donor and acceptor and intron sequences, and termination/polyadenylation sequences. Transgenes must be excised from the bacterial plasmid sequences in order to be expressed in mice.

We inject the linearize DNA into fertilized eggs; those eggs that survive are then implanted in foster females to develop to term. The pups, one week before their wean, will be transferred out of the facility to the care of the investigator upon weaning. The investigator must purify DNA from the tail biopsies and analyze the samples to determine which mice carry the transgene. The minimum elapsed time between injection of the construct and readiness for breeding of the transgenic founders is 9 to 11 weeks.

Several factors can affect the production of transgenic mice. For example, the purity of the DNA construct to be injected is very important to avoid toxic effects to the eggs, and the DNA must be at the proper concentration. The DNA construct must be purified away from vector sequences. Also, some expression constructs may have activities that are lethal for the transgenic embryos. Another caveat is that if transgene integration occurs after the first round of DNA replication in the single fertilized mouse egg, the mouse will be mosaic for the transgene, i.e. only a subset of cells will carry the transgene, and if the germ cells don’t carry the transgene, it will not transmit to the offspring. We will make every reasonable effort to make transgenic mice with your construct, but no facility can guarantee success with every construct. If the studies on tail biopsy DNA (with adequate controls) fail to demonstrate the presence of transgenic animals, we will reinject the construct at no additional cost to the investigator. If no transgenic animal is detected after the second set of injections, we will hold a meeting to discuss further plans, such as re-purification of the DNA construct, or repeat injections and analysis of potential transgenic embryos if the DNA construct may result in embryonic lethality.

We can cryopreserve sperm or embryos from your mice.

In order for us to use your males for either sperm or embryo cryopreservation, they will have to be transferred to our animal protocol and room, but the per diem will be charged to your account. You should request for mouse transfer via CCM.

You should test adult (3 to 6 months) homozygous (preferred) males to determine their fertility. Using experienced males who have already impregnated females and rested for two weeks gets the project off to a quick start.

For sperm cryopreservation, we need two males between 3-6 months of age with proven fertility and separated from females for two weeks. Older males can be accepted in case there is no alternative. We’ll perform a test thaw after freezing and store minimal 10 straws per line.

For embryo cryopreservation, we normally purchase on your account females of an appropriate strain to present to your males, resulting in heterozygous embryos for freezing.  If your strain requires that we use your females, you should be able to provide 3 week-olds, one per male, for 12-18 females depending on how many embryos you want to freeze.  We will superovulate the females and present one female to each male once a week, then harvest embryos for the freezing procedure until the requested number of embryos has been preserved. We will thaw one straw to check for viability and we will either return the males to you or euthanize them per your instruction.

We can revive mouse lines with frozen sperm or embryos from in-house stored materials or shipped from external sources.

When you import sperm from external sources, please work with CCM for importation paperwork and shipment arrangement. Please keep us informed so that we know when to expect the shipment. We’d prefer to handle the transfer of the frozen stock from the shipper to our liquid nitrogen tank. You should ask the source to provide us with a rederivation protocol.

This training course, with combination of lectures and hands-on exercises, is intended for those who wish to develop an expertise in the latest CRISPR and classical transgenic technologies and applications.
Topics/Activities:
Overview of genetically engineered mouse models
Introduction to the CRISPR technology
Gene editing strategy and design for your project of interest
CRISPR tools and available resources
Editing reagent delivery into cells and embryos
Detection and analysis of CRISPR-induced genomic modifications
Interactive discussion on best practices
Tour of the GEMM facility with observation and hands-on opportunity