Transgenic/Knock-out Mouse - Services
Production of Transgenic mice: TG/KO provides services for the generation of transgenic mice through pronuclear microinjection of conventional transgene constructs as well as BACs (bacterial artificial chromosomes). Specifically, TG/KO offers the technical services including (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 utilizes the inbred FVB/N and F1 (C57Bl6/JxCBA/J) strain for the generation of transgenic mice; however, microinjection into the C57Bl6/J strain is also offered.
Interested investigators complete a request form prior to initiation of any service and indicate their level of familiarity and expertise with transgenic techniques. 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 are 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 services include (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 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, W4, W9.5 (all derived from 129/Sv substrains), Bruce 4 (C57Bl6/J) and F1 hybrid lines (C57Bl6/J-129Sv).
Interested investigators complete a request form and consult with Dr. Bhaumik to discuss the overall targeting strategy and experimental design. More extensive consultation with the Drs. Kang and Bhaumik is also 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: TG/KO performs ancillary molecular biology tasks to support and expedite the generation of transgenic and knock-out mice. As noted above, TG/KO 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 knock-out 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. TG/KO also offers genotyping services to investigators in their propagation and breeding of mouse lines.
Consultation on Mouse Husbandry, Breeding, and Phenotypic Analysis: Because many CINJ investigators may be relatively unfamiliar with the handling and use of mice, TG/KO 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, TG/KO attempts to guide investigators in appropriate directions for their ongoing experiments. The resource also interfaces 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 primarily as 8-cell embryo and sperm cryopreservation. Early on, it became clear that there was sufficient demand for storage of lines that are difficult to store through sperm cryopreservation (primarily those in an inbred C57Bl6 and 129Sv background), so TG/KO also offers embryo cryopreservation.
Strain Re-derivation: TG/KO 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.