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Transgenic/Knock-out Mouse

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Services

Production of transgenic mice: The Resource provides services for the generation of transgenic mice through pronuclear microinjection of conventional transgene constructs as well as BACs (bacterial artificial chromosomes). Specifically, the Resource offers the following technical services: (1) preparation of DNA for microinjection using constructs provided by the investigator; (2) superovulation and mating of mice to generate fertilized zygotes; (3) pronuclear microinjection of DNA into zygotes; (4) surgical re-implantation of injected zygotes into pseudopregnant female hosts; and (5) screening of progeny mice for transgene incorporation by Southern blotting of tail tip DNA. The Resource will initially utilize the hybrid F1(C57Bl/6xCBA) strain for the generation of transgenic mice; if there is sufficient investigator interest, microinjection into the FVB, C57Bl6/J strain may also be available.

Consultation is available for investigators who are inexperienced or who wish to attempt more advanced techniques (e.g., BAC transgenics). In addition, ancillary molecular biology services is available for the preparation of transgene DNA constructs for microinjection, as well as for the genotyping of potential founder mice (see below). These services are provided on a fee-for-service basis with guarantees for the number of zygotes injected, as well as founders to be obtained if the DNA is prepared by the Resource. In the event of failure to generate the guaranteed number of founders, the investigator will only be charged for the cost of mice and reagents.

Production of gene-targeted mice: The Resource provides a comprehensive service for the generation of conventional gene-targeted (“knock-out”) mice, incorporating ES cell culture, gene targeting, and generation of chimeric mice by blastocyst injection. Specifically, the Resource provides the following services: (1) culture and electroporation of ES cells; (2) selection, picking, and freezing of ES clones; (3) genomic DNA preparation from ES cell clones and screening for targeting events by Southern blotting; (4) mating of mice to generate blastocysts; (5) injection of targeted ES cells into blastocysts; (6) surgical re-implantation of injected blastocysts into pseudopregnant female hosts; (7) mating of high-grade male chimeras; and (8) screening of progeny mice for germline transmission by Southern or PCR of tail tip DNA. Investigators have the option of providing targeting constructs to the Resource for electroporation into ES cells, or instead providing targeted ES clones that have been generated in their own laboratories for subsequent injection to generate chimeric mice (steps 4-8). Several different ES lines are available for gene-targeting experiments, including TC1, R1, W9.5 (all derived from 129/Sv substrains) and Bruce 4 (C57Bl6/J); in addition, F1 hybrid lines (C57Bl6/J-129Sv) will soon be available.

Interested investigators should complete a request form and consult with the Resource Manager to discuss the overall targeting strategy and experimental design. More extensive consultation with the Manager and Director is available for investigators who wish to generate conditional or other complex alleles. Molecular biology services are available for the screening of selected ES cell clones by Southern blotting for targeted events, as well as for the genotyping of progeny of germline chimeras for transmission of the targeted allele (see below). These services are provided with "guarantees" for the number of ES cell clones screened for targeting, as well as the number of chimeras to be obtained if the ES cells are generated by the Resource.

Molecular biology services: The Resource will perform ancillary molecular biology tasks to support and expedite the generation of transgenic and knockout mice. As noted above, the Resource can assist with the generation of transgenic mice through preparation of DNA constructs for microinjection, as well as by screening progeny for potential founders. Similarly, the Resource can assist with the generation of knockout mice in screening ES cell clones for targeting events by genomic DNA preparation and Southern blotting, as well as in identification of germline transmission by PCR of tail tip DNA. Depending on investigator demand, the Resource may also offer genotyping services to investigators in their propagation and breeding of mouse lines.

Consultation on mouse husbandry, breeding, and phenotypic analysis: Since many CINJ investigators may be relatively unfamiliar with the handling and use of mice, the Resource offers services including: (1) training in the proper use of a barrier facility; (2) handling and breeding of mice; (3) maintenance of breeding records; (4) weaning, ear-tagging, and tail tip cutting; (5) mouse and embryo dissection; and (6) basic phenotypic analysis of adult and embryonic stages. While it is impractical to offer every aspect of phenotypic analysis as a service, the Resource will attempt to guide investigators in appropriate directions for their ongoing experiments. The Resource will also interface with the Biometrics Shared Resource to assist investigators in determining the optimal numbers of mice to be utilized in their experiments to obtain statistically meaningful results.

Mouse strain cryopreservation: Complementary to the tasks of generating new mouse strains, the Resource also provides services for the long-term storage of inactive lines. The Resource primarily focuses on sperm cryopreservation, since this technology does not require expensive freezing equipment, and can be performed with a minimum number of mice. If there is sufficient demand for storage of lines that are difficult to store through sperm cryopreservation (primarily those in an inbred C57Bl/6 background), the Resource will also offer embryo cryopreservation.

Strain re-derivation: The Resource offers strain re-derivation to accommodate two common situations. First, CINJ investigators may be holding existing mouse stocks in “dirty” facilities, e.g., exposed to MHV (mouse hepatitis virus), pinworms, mites, and less virulent pathogens such as parvovirus. Second, mice imported from external facilities will often have health profiles that do not permit introduction into barrier facilities. In both cases, the Resource offers re-derivation by embryo transfer as a service for interested investigators.