Transgenic Mouse Core

The Transgenic Mouse Core Facility is a state-of-the-art resource operated through the Division of Endocrinology to serve the University of Arkansas for Medical Sciences (UAMS), Central Arkansas Veterans Healthcare System and Arkansas Children’s Hospital. The Core consists of dedicated laboratory space with a microinjection suite, cryopreservation lab, and isolator rack animal wards.

Additional Information
Services Provided & Fees
Technology Description
Protocols
Request Services
Contact Information

Overview of Core Services

Services Provided and Fees

  • Transgene Design Consultation
    There is no fee for this service.
  • Genotyping Assay Design Consultation
    There is no fee for this service.
  • Transgenic Mouse Production With Inbred or Hybrid Strains
    The Transgenic Mouse Core Facility generates transgenic mice via pronuclear microinjection of DNA constructs supplied by users. Microinjections are performed in embryos obtained from C57BL/6 or CB6F1 (a cross between BALB/c and C57BL/6) mice. After weaning potential founder mice, a small amount of tissue is taken from the tip of the tail and provided to users for genotyping. At that time each individual pup is permanently identified with an ear punch corresponding to the number it is given in the Transgenic Mouse Core Facility database. Users then identify mice harboring the transgene, usually by PCR amplification. To avoid costs, users must identify founders and initiate transfer of mice within three weeks. After that period, users will be charged a per diem rate of $2 per cage, per day. Please also note that production of transgenic mice will not be initiated unless an approved IACUC protocol is in place to allow transfer of the mice once they are identified.The core assesses a service fee of $3,200 for the generation of at least two transgene-positive mice.
    • Sperm Cryopreservation

      When a mouse strain is not immediately needed, but may be needed later and cannot be readily obtained from an approved vendor, mouse sperm cryopreservation provides an efficient solution for backing up many genetically engineered mouse strains. Sperm cryopreservation can also protect mouse strains that are difficult to replace in the event of a pathogen outbreak, natural disaster, or cessation of your existing breeding stock. Sperm is isolated from two male mice between the ages of 3-6 months. Ideally, these would be males that have bred successfully in the past, but separated from the female for at least two weeks. Sperm is isolated and treated with a cryoprotective media that enables the germplasm to be stored indefinitely in liquid nitrogen. Because of the strain-to-strain variation in genetically engineered mice, we cannot always guarantee the success of this procedure. As a means of quality control, however, we routinely cryopreserve a test-straw to evaluate the viability, mobility and concentration of the frozen sperm. We are now including in the cost of sperm cryopreservation an additional step of quality control. We will use sperm from the test-straw to fertilize eggs and culture these embryos to the two-cell stage. If the percentage of eggs that go on to two-cell embryos is low, the investigator will be notified.

Sperm cryopreservation has three significant biological disadvantages compared to embryo cryopreservation:

      1. Since a haploid genome is being frozen, more breeding of reconstituted mice may be necessary if more than one mutation and/or transgene is involved.
      2. Some mutations must be maintained on a mixed or otherwise unusual background to maintain the desired phenotype, and oocyte donors with that background may not be available at the time of reconstitution.
      3. As mitochondria are inherited exclusively from the maternal lineage, cryopreservation of sperm cannot be used to preserve strains of mice with mtDNA polymorphisms.

The core assesses a service fee of $650 for sperm cryopreservation. Long-term storage in liquid nitrogen is also included in this fee.

  • In Vitro Fertilization
    The reconstitution of live mice from cryopreserved sperm is accomplished via in vitro fertilization (IVF). This service includes the purchase price and super-ovulation of the egg-donor females, providing pseudopregnant foster dams for uterine transfers, per diems for pregnant embryo recipients, as well as the weaning and transfer of pups to the investigator. The core assesses a service fee of $650 for in vitro fertilization.
  • Rederivation
    Rederivation is used to help eliminate pathogens from a mouse line. This may be required by the Division of Laboratory Animal Medicine if the health status of a desired strain is in question prior to its importation from another facility, or if a strain tests positive for a pathogen and elimination of the colony is the only other solution. Rederivation is accomplished by harvesting embryos from a group of females provided by the investigator. The isolated embryos are taken through a series of washes and then transferred into pseudopregnant foster dams. The foster dams are quarantined until their pups are weaned, and then testing is performed on the mothers to determine if the pathogen has been eliminated. The core assesses a service fee of $600 for rederivation. This includes the isolation and transfer of the embryos but does not include special fees for quarantine or the serological testing of the mothers.

Technology Description

The transgenic mouse has assumed increasing importance as an experimental in vivo model in recent years. The uses of this technology are numerous, but include studies of normal and altered gene function, analysis of elements responsible for developmental and tissue-specific gene expression, and the production of animal disease models.

Transgenic mice are generated by injection of a recombinant DNA construct directly into the male pronucleus of a fertilized egg. The injected eggs are transferred to pseudopregnant females and the resultant pups are genotyped for the presence of the transgene by PCR amplification of DNA from tail biopsies. Each transgene positive founder represents a unique integration site which may affect transgene expression or regulation.

An individual transgene construct is injected with the goal of producing up to 30 potentially transgenic pups. Tail DNA is then provided to investigators for analysis. Once transgene positive pups are identified, they are transferred to the investigator for use.

transgenic mouse core

Protocols

Plasmid DNA for Microinjection

Submit at least 1 µg of linear gel-purified DNA for microinjection. The DNA should be in H2O or TE at a concentration of at least 20 ng/µl. Several methods of extraction from the agarose gel will provide DNA suitable for transgenic mouse production; however, some methods are better than others. We routinely use the Zymoclean Gel DNA Recovery Kit from Zymo Research (Catalog number D4001). Qiagen and Elutip kits have worked as well. The quality of the plasmid DNA preparation is also important. We recommend use of the Qiagen Endotoxin-free kit. An extensive discussion of DNA purification for microinjection can be found in Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press.

We will check the concentration and size of DNA in the sample, dilute it in microinjection buffer, and sterilize it by filtration. Samples of insufficient quantity or concentration, or samples with degraded DNA or excessive amounts of agarose will be returned to the investigator.

Last update: 1/31/12

Preparation of Positive Control Templates for Genotyping PCR

  1. Determine ratio of transgene to mouse genome by dividing 3×109 bp by the size of the transgene in bp.

    For example, if your transgene is 5 kb long then: 3×109 ÷ 5000 = 6×105

  2. Determine the amount of transgene DNA required to equal the equivalent of 1 copy per genome in 200 ng of genomic DNA. 200 ng is the standard amount of mouse genomic DNA used in a genotyping reaction.
    Continuing the example above:
    200 ng/x = 6×105/1
    x = 0.00033 ng
    Therefore, you would need to add 0.00033 ng of your 5kb transgene DNA to 200 ng of mouse genomic DNA to create a positive control template that contains the equivalent of a single copy transgene insertion.Also prepare templates containing the equivalent of 5 and 10 copies of the transgene per genome.
    In the example:
    5 copies = 0.00033×5 = 0.0017 ng
    10 copies = 0.00033×10 = 0.0033 ng
  3. Prepare the necessary dilutions of your transgene DNA so that the appropriate amount of transgene DNA can be added to the 200 ng of mouse genomic DNA in 1 microliter. Then perform the PCRs to determine whether the PCR assay is sufficiently sensitive to detect the 1 copy/genome control. If it cannot, adjust the PCR conditions or redesign the primers.

Request Services

Sperm Cryopreservation Work Order Request
In Vitro Fertilization Work Order Request
Transgenic Mouse Work Order Request

Contact Information

Transgenic Mouse Core
Winthrop P. Rockefeller Cancer Institute, Room 834
4301 West Markham Street
Mail Slot 587
Little Rock, AR 72205

Charles O’Brien, Ph.D., Director
Department of Internal Medicine
UAMS College of Medicine
caobrien@uams.edu
501-686-5607

Joseph Goellner, M.S., Managing Director
jjgoellner@uams.edu
501-940-7264