The purpose of the Transgenic & Chimeric Mouse Facility is to provide a centralized service to efficiently produce transgenic mice for basic research. This should result in reduction in effort and cost to participating investigators. The facility is located on the basement level of the Clinical Research Building. This facility consists of an animal room and several injection rooms. The injection rooms are fully equipped to carry out the entire procedure of making transgenic mice. The animal room provides housing and breeding space for the mice involved in the transgenic projects. The facility uses sterile food and water as well as autoclaved cages and bedding; all cages are of the microisolator type to limit the spread of colony infection. The entire facility is located behind a microbiologic barrier where admittance is strictly limited and all personnel must wear sterile coveralls, gloves, hats, masks, and boots.
Richa, Jean., PhD
Role: Adjunct Professor, Department of Genetics, Technical Director, Transgenic & Chimeric Mouse Facility
Two recommended procedures are provided here for your convenience. Each has been thoroughly tested and are equally effective in producing transgenic mice using DNA constructs.
Modification of Dr. Patricia Labosky's protocol. Courtesy of Raluca Verona from the laboratory of Dr. Marisa Bartolomei, Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine.
Procedure for preparation of a sample of ES cells for injection into blastocysts.
Courtesy of Joanne Thorvaldsen from the laboratory of Dr. Marisa Bartolomei, Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine.
Morrisey Lab Protocol, by Dan Swarr & Dave Frank
Protocol for isolating high-molecular-weight DNA from mouse tails (from Manipulating The Mouse Embryo; Hogan et al., Cold Spring Harbor, 1986).
"Since all animal maintenance and subsequent breeding of transgenic mouse lines will be carried by the individual investigator, a knowledge of basic mouse husbandry is essential. Here are some basic guidelines and suggestions."
Courtesy of Dr. Douglas Epstein, Department of Genetics, University of Pennsylvania School of Medicine.
Protocol for the correct preparation of YAC DNA as well as for the large scale preparation of yeast plugs and various related solutions are provided here.
Courtesy of Dr. Jorge Henao-Mejia, Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania.
Introduction of DNA and/or RNA into fertilized oocytes
(Description coming soon)
"Direct microinjection of DNA into the male pronucleus of a mouse zygote has been the method most extensively used in the production of transgenic mice. If the foreign DNA integrates into the mouse chromosomal DNA at the one-cell stage, the animal will contain the injected DNA in every cell, including those of the germ line. If, however, integration occurs later, the resulting animals will be mosaic for the presence of the injected DNA. The number of copies of integrated DNA can vary from one to several hundred, arranged primarily in tandem head to tail arrays.
What the Facility will Provide
Upon receipt of the DNA construct, proven by documentation to be of high quality and suitable for microinjection (see below), we will proceed to inject a minimum of 150 fertilized eggs. After overnight incubation, normal 2-cell embryos will be transferred into foster females and their pregnancy will be monitored (the gestation period of the mouse is 19-20 days). Two weeks after birth of potential transgenic pups, the project scientist will be notified of the number of pups.The facility will carry out tail clipping, animal tagging and genotyping by PCR according to the conditions set by the project scientist. At weaning, the positive founders will be transferred to the care of the individual investigators as per ULAR procedures.If these studies (with adequate controls) fail to demonstrate the existence of at least one transgenic animal, we will reinject at least 80 eggs at no additional cost to the investigator. If, however, no transgenic animal is detected after the second trial, a meeting will be held between the scientist, the Facility Co-Directors, and the Technical Director to discuss the DNA construct, the tail biopsy analysis, and further plans to be agreed upon. On average, 230 injected eggs should yield at least one (and usually more) transgenic mouse."
"Freezing mouse embryos is a convenient way to preserve in important mouse line for possible future use or to protect this line against loss due to infection, lab accident, etc. Embryo freezing could also result in substantial reduction in housing cost and cage use.
A set of five to seven young male mice will be needed to generate enough embryos for each freezing set. The male founders will be subjected to a waiting period in the containment area, as required by ULAR, prior to admission into the barrier colony.
A minimum of 300 healthy embryos will be frozen per transgenic/knockout line that is hemizygous for the transgene. In the case where the mouse line is homozygous for the transgene, 150 embryos will suffice. Several weeks after the completion of the freezing process, a few embryo samples will be removed and processed through thawing and monitoring development to assess their viability and ability to develop to term. The facility will now store the frozen samples and maintain the records of your cryopreserved lines."
"The production of chimeric mice by introducing Embryonic Stem (ES) cells into early embryos is an alternative approach to generating transgenic mice. The technique utilizes the tissue culture system to modify and select ES cells that had received an exogenous DNA of interest. Once derived and characterized, ES cell clones may be transferred into 4-day-old mouse embryos where they can differentiate into any adult tissue type.
What the Facility will Provide
Through this service, the facility will generate chimeric mice by injecting Embryonic Stem (ES) cells containing site specific genomic alterations (knockouts) into normal blastocysts. ES cells will be injected into a minimum of 50 blastocysts of C57BL/6J or Balb/c. When the blastocysts have reexpanded, they will be transferred into the uterine horn of pseudopregnant female and allowed to go to term. Upon weaning 3 weeks after birth, chimeric mice are separated and then released to the care of the investigator."
"Through this process, a mouse line that faces reproductive obstacles can be rescued, provided that the fertilizing efficiency of the sperm is maintained and that a compatible egg donor strain is available.
This service can be provided simultaneously with sperm cryopreservation in an effort to re-derive the mouse line while securing frozen samples for storage.
The details of this service will be tailored to the needs of individual investigators."
"This process involves the surgical transfer of embryos into pathogen-free recipient females, where they can develop to term. Rederivation is the most effective method to rid a colony of pathogenic infections. It is also recommended when genetically altered animals are acquired from outside institutions that do not fall into the category of approved vendors. With the proper procedure, mouse lines can be saved or transferred safely across distant locations.
Due to the diverse environmental conditions among the animal facilities located across campus, the structure of this service will be tailored to fit each and every facility receiving the rederived line. As a consequence, the fees may vary accordingly. Basically, a total of 50 embryos generated from the designated mouse line will be transferred into recipient pathogen-free females that would carry them to term in a housing location assigned by ULAR. The mice born from such transfers should be of sufficient number to rejuvenate or establish the line."
Alternative service for re-derivation: IVF
"The epididymis of the donor male will be harvested by the user (as in 1., above), delivered to the TCMF, and the released sperm will be used for IVF of wild-type oocytes. The oocytes will have to be harvested in the TCMF from females acquired from approved vendors. Fertilized oocytes will be cultured overnight and any resulting 2-cell stage embryos will be transferred into surrogate females. The transplanted pseudo-pregnant recipients will then be then transferred to the Levy building for gestation and quarantine monitoring. ULAR diagnostics must be contacted and space within the Levy quarantine cleared before any date for IVF can be set. The user should consult with ULAR regarding duration and rules of quarantine in Levy."
Alternative service for re-derivation: Embryo transfer
"This requires hands-on training of lab personnel to perform intraperitoneal (ip) hormone injection and to dissect uterine horns. All media and hormones will be supplied by the facility as will the actual training. The user’s lab will be responsible for two ip hormone injections, 48 hrs apart between the hours of 12 PM and 2 PM, into a set of 3-week old females. Once the second hormone injection is complete the female mice should be housed with single caged males and left alone for 3.5 days. On the morning of harvest, the females will be sacrificed by the user, uterine horns removed and a lab member who has not been near the actual animals must deliver the organs to the TCMF (behind the microbiological barrier) in doubled Petri dishes. ULAR diagnostics must be contacted and space within the Levy quarantine cleared before any date for embryo harvest can be set. The user should consult with ULAR regarding duration and rules of quarantine in Levy."
"This service provides an alternate procedure to preserve important mouse lines that either fail to generate embryos for cryopreservation, or when the breeder males are not located within the barrier Facility. It offers similar benefits to those of embryo cryopreservation by protecting the mouse lines from losses and by reducing the housing cost and cage space.
For this service we recommend a one-time harvest of embryos to be frozen prior to sacrificing the males for cryopreservation.
A set of 5-7 young male mice will be subjected to a waiting period in the containment area, as required by ULAR, prior to admission into the barrier colony. Once admitted, the animals will be mated with females to generate embryos to be frozen. Then 3 males will be sacrificed and the sperm collected and frozen."
Alternate service for sperm cryopreservation
"The user isolates the epididymis from 3 healthy males and brings them to the TCMF. The sperm will be isolated and frozen. The TCMF can bring back these lines subsequently via in vitro fertilization (IVF). For sperm cryopreservation at least 3-5 males between the ages of 8 weeks and 6 months are optimal. For animals that cannot be transferred to the CRB facility, the isolation must be done by the user in his/her own facility. This requires hands-on training of lab personnel for the epididymis harvest. Media will be supplied by the TCMF to all labs. Five straws of sperm per male will be frozen. For this service , we recommend a one-time harvest of embryos to be frozen prior to sacrificing the males for sperm cryopreservation."