 |
|
|
|
Transgenic Mouse Service |
|
|
|
|
|
 |
||
The Transgenic Mouse Service (TMS) creates transgenic mice by pronuclear injection of (C57BL/6J X SJL/J) F2 embryos with transgenic expression cassettes prepared by investigators. Creating a transgenic mouse requires several critical steps.
Each of these steps requires coordination between investigators and the TMS. Please read the following information before submitting your construct: Transgenic expression cassettesTransgenic expression cassettes that use genomic DNA rather than cDNA are generally more efficiently expressed in mice. Consider including generic introns when cDNA is used and levels of expression are important. Select promoter sequences with proven tissue specific expression in transgenic mice where possible. Proper purification of transgenic expression cassettesProper purification of transgenic expression cassettes is crucial for successful transgenic mouse production. Mouse embryos are highly susceptible to trace contaminants such as phenol, ethanol, endotoxin, and enzymes. In addition, particulates obstruct microinjection pipettes. The number of live births from microinjected embryos is a direct function of the purity of the DNA being microinjected. Transgenic expression cassettes must be excised from highly purified plasmids, isolated by sucrose gradient centrifugation or electrophoresis followed by electroelution, and the linearized isolated fragments further purified using one of several protocols. If DNA submitted is deemed to be inferior based on results of pronuclear injection and embryo transfer, the investigator will be charged and asked to submit a new sample. BAC TransgenicsThe TMS will microinject BAC DNA to create the desired transgenic mice. However, due to the size of such constructs, the time between submission and microinjection of the DNA presents a potential problem for stability of the BAC DNA during storage. In addition, high purity of the DNA is absolutely critical but tricky to achieve; extra care must be exercised in order to prepare BAC DNA that can be microinjected. With these factors in mind, the TMS recommends that prior to construct preparation, users refer to the website of the University of Michigan Transgenic Animal Model Core for protocols for DNA preparation and storage. In particular, note the use of polyamine buffer for “long-term” storage of purified BAC DNA that may obviate the need to submit DNA that is prepared within 24 hrs. of microinjection. In addition, one of our Yale users, Dan Kaplan, recommends that if a gel is used to purify the BAC, the molten agar should be filtered through a 0.22uM filter prior to casting to remove impurities that may block microinjection needles and prevent the DNA from being microinjected. The problem of DNA stability causes problems with scheduling of BAC microinjection. Due to the need to schedule microinjection dates in advance (as opposed to adding the construct to the queue of traditional constructs), and the corresponding need to order the appropriate mice, carefully prepared DNA must be submitted to TMS as scheduled. If such a scheduled arrangement is necessary, and the investigator is unable to submit the DNA as planned, the investigator will be charged for the mice ordered and used for that date. Pronuclear microinjectionPronuclear microinjection of approximately 250 embryos of (C57BL/6J X SJL/J) F2 mice is performed typically over four days; 200 to 250 are transferred to the uteri of six pseudopregnant foster mothers. Litters are maintained by the TMS pending results of tail genotyping. Genotyping miceInvestigators are provided tail biopsies from individually identified weanling mice six weeks after embryo microinjections are completed. Investigators extract DNA from tail biopsies using established protocols and identify individuals with integrated sequences by polymerase chain reaction (PCR) analysis or Southern blot analysis. After positive mice are identified, an animal relocation request form is completed so that mice can be transferred to the investigator’s animal room(s). Breeding transgenic micePotential founder mice become the property of the investigator
who may wish to obtain additional advice and services related to
breeding from the YARC Rodent Service. Animals that test positive
for integration of transgenes may have copies in all somatic and
germ cells or may be mosaic. It is important to note that integration
into the genome is random so that each transgenic animal is uniquely
hemizygous. The genome of each potential founder must be reiterated
by mating with non-transgenic mice to demonstrate germline transmission
and to select hemizygous individuals for intercrossing to produce
homozygous transgenic lines. Transgenic Mouse Service Order
Form Contact James McGrath, M.D., Ph.D. (785-2686) or Carole Pease (785-6923). |
