Transgenic Mouse Core Facility

Introduction
WMC now offers Transgenic Mouse Core services, as a joint effort with Sloan-Kettering Institute (SKI). This Facility, which is under the direction and guidance of Dr. Elizabeth Lacy at SKI, will allow WMC investigators access to many of the services offered at SKI's Transgenic Mouse Facility. A WMC transgenic core oversight committee will be responsible for periodic review of the quality and access to this service.

Location
1209 Rockefeller Research Laboratory at SKI: 639-8907
1211 Rockefeller Research Laboratory at SKI: 639-8907
1206 Rockefeller Research Laboratory at SKI: 639-8906
937 Rockefeller Research Laboratory at SKI: 639-8661
917A Rockefeller Research Laboratory at SKI: 639-8906

Staff
Elizabeth Lacy, Ph.D., Head
Willie H. Mark, Ph.D., Manager
Jia-Hiu Dong, M.D., Research Assistant
Joanne Ingenito, B.S., Research Assistant
Bo Wang, B.S., Senior Technician
Rona Lester, B.S., Senior Technician
Anthony Zayas, Supervisor, Special Husbandry Services

Facility Description
This Facility produces transgenic and chimeric animals using, respectively, DNA constructs and embryonic stem (ES) cells provided by the investigator. It also cryopreserves embryos from transgenic, gene targeted, and congenic lines. Physically, the Facility consists of three animal rooms that house the stock mice required for the production of transgenic and chimeric animals; one room (RRL 1211) containing three microinjection stations, a pipette puller, and a CO₂ incubator; one room (RRL 1209) containing stereomicroscopes for the isolation and surgical transfer of mouse embryos, a CO₂ incubator, and microforge; one room (RRL 1206) equipped with a single microinjection station, a pipette puller, and a RTS Systems BC-1 Controlled Rate Freezer; one room (RRL 937) for ES cell culture, equipped with a two laminar flow hoods, two double chamber CO₂ incubators, and a Nikon TMC tissue culture microscope. All microinjection stations are equipped for the injection of DNA into eggs and the injection of ES cells into blastocysts.

Organization and Services Provided

1. Consultation

2. Transgenic Mice

The investigator purifies the DNA construct for microinjection into eggs; approved DNA purification protocols are provided by the Facility. Before DNA is accepted and scheduled by the Facility for injection, the investigator must demonstrate that he or she has a working PCR or Southern assay for the identification of transgene positive mice. In addition, before scheduling, the investigator must provide the DNA along with a photograph documenting its purity and concentration. If construction of transgenic mice with a given transgene is not covered by an approved animal protocol, a protocol must be submitted to the IACUC and receive approval prior to DNA injection. Contact the IACUC at 746-6079 or visit the IACUC webpage for additional information.
The Facility staff performs the injections of transgene DNA constructs into one-cell fertilized mouse embryos, and the surgical reimplantations of the manipulated embryos into the oviducts of day 1 p.c. pseudopregnant foster mothers. Approximately 300 injected eggs are transferred to pseudopregnant recipients for each transgene construct. If fewer than two transgene positive founder mice are generated, the Facility will perform a second series of injections and transfers at no additional cost.

The Facility performs DNA injections into fertilized one-cell embryos derived from matings between (C57BL/6J x CBA/CA)F1 males and females. However, if required for an investigator's experiments, the Facility will perform DNA injections into eggs bearing a different genetic background. In such cases the user purchases or provides the male and female mice for the matings to generate fertilized eggs.

The staff prepares tail biopsies from the mice that develop from injected eggs, the founder generation. The investigator is responsible for screening these mice for the presence of the injected DNA. Once transgenic founders are identified, they become the financial and scientific responsibility of the investigator, are removed from the SKI Facility, and are transported to the WMC animal facility for long term housing.

3. Chimeric Mice

For blastocyst injections, the investigator prepares, clones, and cultures the gene targeted ES cells. The Facility maintains frozen down aliquots of different ES lines that it obtains from SKI and other institutions. These cell lines are available to all users of the Facility. Currently the Facility has aliquots of CJ7, J1, and R1.

If the ES cell line is not obtained from the core facility, the line must be MAP tested by RARC to confirm the line is free of adventitious viruses and mycoplasma, before providing clones to the Core for injection. Cost for MAP testing is not included in the fee for generating chimeras. Contact RARC's Animal Health Service at 746-1079 for additional information and to schedule testing.

The Facility also provides pregnant neo^r mice for the preparation of embryonic fibroblast feeder layers. For new ES cell users, the Manager provides training in the preparation of feeder layers, in ES cell culture, and in the selection of targeted ES cell clones.
Before a gene targeted ES cell clone is accepted and scheduled by the Facility for injection into blastocysts, the investigator must provide Southern or PCR data demonstrating that the targeting vector homologously recombined into the target locus. In addition, a protocol must be submitted to the IACUC and receive approval prior to ES cell injection. Contact the IACUC at 746-6079 or visit the IACUC webpage for additional information. The investigator should inform the Manager of the Facility when the electroporation component of the targeting experiment is initiated. This allows the Facility to anticipate and plan for the workload about a month in advance.

The Facility staff performs the injections of ES cell clones into mouse blastocysts and the surgical reimplantations of the manipulated embryos into the uteri of pseudopregnant foster mothers. Approximately 60 injected blastocysts are transferred to pseudopregnant recipients for each ES cell clone.

The Facility performs ES cell injections into blastocysts derived from the C57BL/6J inbred strain. However, if required for an investigator's experiments, the Facility will perform ES cell injections into blastocysts bearing a different genetic background. In such cases, the user purchases or provides the male and female mice for the matings to generate the blastocysts.

The Facility staff breeds all chimeras to C57BL/6J mice to monitor for germ line transmission of the ES cell genome. Identified germ line transmitting chimeras and their progeny become the scientific and financial responsibility of the investigator's lab. The animals are removed from the SKI Facility and transported to the WMC animal facility for long term housing.

4. Embryo Transfer Rederivation for Strain Importation

Embryo transfer (ET) has been demonstrated to be an effective method to rederive mice known to be infected with adventitious viruses, parasites, and bacteria. ET rederivation must be coordinated with both the Research Animal Resource Center (RARC) and the WMC Transgenic Mouse Core facility. RARC should be contacted for details pertaining to mouse importation and to coordinate shipment and receipt of the animals to be rederived.

Mice cannot be brought to WMC without first obtaining RARC's approval. Receipt of frozen embryos is preferred to the importation of live mice. In brief, approximately 50 frozen embryos or a minimum of two male mice of the imported strain are required. If importing frozen embryos, the embryos will be thawed and surgically transferred into recipient mice.

If live mice are imported, they will be bred to superovulated females of the desired strain. The investigator provides or purchases females of the appropriate strain for superovulation and mating. These mice must be three weeks of age in order to obtain an optimal response to superovulation.

Subsequently, embryos are harvested and transferred into recipient mice. All surgical procedures are performed by the Facility personnel in WMC procedure rooms. At weaning, the recipients and offspring are tested by RARC to ascertain they are free of adventitious viruses, parasites, and pathogenic bacteria. If they are negative to the agents in these agents, the animals are released to the investigator for their use.

5. Cryopreservation of Transgenicm, Gene Targeted, and Congenic Mouse Strains

The investigator provides the Facility with homozygous or heterozygous males for each strain to be cryopreserved. The male mice are mated to superovulated 3-4 week old females to provide embryos for freezing. The investigator purchases or provides the female mice for superovulation. These must be three weeks of age in order to obtain an optimal response to superovulation. If not from the transgenic, gene targeted, or congenic strain to be cryopreserved, the superovulated females are generally of the C57BL/6J or the (C57BL/6J or CBA/CA) F1 strain background.

Eight cell morulae are recovered on day 2.5 p.c. and cryopreserved in straws using a two step freezing procedure and a RTS Systems BC-1 Controlled Rate Freezer. Approximately 300 embryos are cryopreserved per line. One straw is thawed per line to check that embryos can be recovered at an acceptable frequency following oviduct transfer (? 10% of the transferred embryos). If the frequency of recovery is less than 10%, more embryos are collected for cryopreservation.

6. Training

Part II: Fees for Services

DNA Injections: $1,600 per construct

This includes 8-10 oviduct transfers, each with approximately 30 injected eggs. If the first series of injections produces fewer than 2 transgene positive founder mice, the Facility will do a second series of injections at no extra charge. If no or fewer than 2 transgene positive founders are obtained after the second series, the user must meet with Drs. Lacy and Mark before anymore injections are performed with a given construct. There will be a charge of $1600 for a third series of injections.

ES cell injections: $600 per clone

This includes 3-4 uterine transfers, each with 12-15 injected blastocysts. In addition, this fee includes breeding the resulting chimeric mice to assay for germ line transmission of the ES cell genome. To maximize the chances for germ line transmission, investigators usually provide 2-3 independent ES cell clones per gene knockout.

Embryo rederivation¹: $700 per line

This includes superovulation of egg donors, setting up matings, recovery of fertilized eggs, and the surgical transfer of the fertilized eggs to pseudopregnant foster mothers. The investigator is required, however, to purchase or provide females for superovulation.

Cryopreservation: $1,700 per line

Special Services: $40 per hour per staff member

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¹Fees do not include RARC charges for testing ET derived offspring and recipient dams for viruses, parasites, and bacteria; per diem for mice maintained at WMC for breeding to generate embryos; or the purchase of special strains of mice used to generate embryos if these strains are not available in the Core.

Part III: Procedures for Use of the Transgenic Mouse Core Facility

Person to Contact

Investigator Request Forms (Word documents, will download to your desktop)

IACUC Approval

Recombinant DNA

Long Term Animal Housing

WMC's Research Animal Resource Center (RARC) will provide long term housing for all animals derived from the Transgenic Core Facility. Investigators are advised to contact Dr. Scott Perkins, 746-1043, or RARC to arrange space as soon as possible. Approximately 20-30 cages are necessary for the first six months of work on any given transgenic line.

Scheduling

The following fees will be charged for use of the Transgenic Mouse Core Facility as of November 1, 1997 (to be evaluated annually).

Billing
Billing will be generated at the time of delivery of the construct, ES cell clones, or embryos to the Facility. Investigators will be billed directly by WMC-RARC. (SKI will provide WMC-RARC with appropriate billing information)

Part I: Description of Services