Transgenic Core
Beth Israel Deaconess Medical Center
Video courtesy of Joel Lawitts, which shows DNA being injected into a one cell mouse embryo
Director:
Collaborator:
Transgenic Facility Director:
Click here for core presentation on October 18.
The purpose of the Transgenic Core is:
- To provide a service facility that will utilize investigator-derived DNA constructs or investigator derived (or obtained) genetically modified embryonic stem cells to create founder transgenic and gene knockout or knockin mice (i.e mice bearing point mutants, lox-modified or ires/2A-Cre knockin alleles) that can be used to address questions relevant to obesity, diabetes and nutrition, including questions related to brain control of feeding, metabolism and reward.
- To provide consultation and training on all aspects of making and using genetically modified mice.
Core Services
(with “charge-back” fees for BNORC members)
Effective No. 20, 2021 – due to increases in our supply costs we are increasing our charge back fees by a small amount (new prices listed below).
- Generation of Transgenic Mice. DNA transgenic constructs, supplied by the investigator, are microinjected into fertilized one cell embryos. Tails from potentially transgenic founders are then supplied to the investigator for genetic analysis. Identified founders are then provided to the investigator. Transgenes can be injected into FVB/N or C57BL/6 mouse embryos. Other strains are available upon request. Charge Back Fee = $3,194 for FVB transgenics and $4,577 for C57BL/6 transgenics.If repeat injections of a new DNA prep are required, because the prior injections failed to produce transgenic mice due to poor quality of investigator-provided DNA prep, this additional injection will cost $918 )* for FVB/N and $1083 * for C57Bl/6.
- CRISPR and “Easi-CRISPR” generation of gene knockout or gene knockin mice, mice bearing loxed alleles, Cre driver knockin mice, XFP knockin mice, etc. Reagents, including DNA constructs, guide RNAs and CAS9 protein supplied by the investigator, are microinjected into fertilized one cell embryos. The required amount of reagents is: 25ug CAS9, 15ug sgRNA, 3-5ug DNA (100 ng/ul minimum if in solution). Reagents may be presented to the Core in a lyophilized form (preferred), or reconstituted in culture grade water. Tails from potentially modified founders are then provided to the investigator for genetic analysis. Identified modified mice are then provided to the investigator. CRISPR reagents can be injected into FVB/N or C57BL/6 mouse embryos. Other strains are available upon request. Charge Back Fee = $3,194 for FVB transgenics and $4,577for C57BL/6 transgenics. Consultation and questions on all aspects of using CRISPR or Easi-CRISPR to make genetically modified mice is provide by Brad Lowell. If repeat injections a re required, no new reagents will be accepted. A re-injection fee of $918 * for FVB/N and $1,083 * for C57Bl/6 will be applied. Investigators could submit new sgRNAs for a given project and be charged a new prep fee. Any submission of new DNA will be considered a new project.
- Gene Targeting – Generation of Targeted ES cells. Transgene DNA constructs supplied by the investigator, are electroporated into 129 ES cells (W4/129S6) or C57BL/6 ES cells (C57BL/6N-PRX-B6N). To ensure quality of downstream procedures, we require an electrophoresis image with linearized DNA construct(s) and a mass DNA ladder be submitted along with the DNA sample. Up to 72 drug resistant clones are selected, expanded and frozen down for subsequent use. Aliquots of cells from each clone are provided to the investigator for genetic identification of targeted clones. Charge Back Fee = $2,000.
- Gene Targeting – Generation of Chimeric Mice from Targeted ES Cells. Targeted ES clones are injected into 3 day old embryos (blastocysts). Highly chimeric mice are then provided to the investigator for breeding. Targeted ES cell clones to be injected may have been generated by the BNORC Core (as above under #3), by the investigator’s lab, or by various high-throughput ES targeting consortiums (for example, EUCOMM – http://www.knockoutmouse.org/about/eucomm). Charge Back Fee for 1 day’s injection (either 1 or 2 ES clones) into black C57BL/6 embryos = $1,307. Charge Back Fee for 1 day’s injection (either 1 or 2 ES clones) into albino** C57BL/6 embryos = $1,577.
- Cryopreservation of Mutant Mice (embryos)*. Fertilized two cell embryos are cryopreserved. Charge Back Fee = $5.35 per mouse embryo. For a typical project, 100 embryos are frozen.
- Cryopreservation of Mutant Mice (sperm)*. New service starting in December, 2021. The Transgenic Core will retrieve the sperm from the epididymis and cryopreserve approximately 6 straws. One straw will be utilized to perform an IVF with a small number of FVB/N females to ensure that the sperm freezing was successful. Charge Back Fee = $920*
Tips on choosing between the two methods of cryopreserving mutant mice (for questions and/or more detailed advice, contact the Transgenic Facility Director, Joel Lawitts – jlawitts@bidmc.harvard.edu):Cryopreservation of embryos (service #5): Best used when an investigator has a homozygous line. The Transgenic Core provides a protocol to grow and set up a colony, and hormones to superovulate mice. Investigators grow the colony, superovulate and mate the mice and bring the reproductive tracts of the female. We will retrieve and cryopreserve the embryos. We will provide training to cut out the reproductive tract.
Cryopreservation of sperm (service #6 above): Best used when an investigator has a heterozygous line, or when the investigator does not have the time, personnel, or finances to grow and maintain a colony. The Transgenic Core will train the investigator to cut out the tail of the epididymis. The Core will retrieve the sperm, perform an IVF with wildtype females, grow the zygotes overnight and cryopreserve the embryos at the 2-cell stage. - Generating Mice from Frozen Embryos. Embryos (frozen) from outside sources (after clearance from the BIDMC Animal Research Facility) or from the BNORC Transgenic Core Facility will be thawed and transferred into pseudopregnant recipients. Charge Back Fee = $606
- In Vitro Fertilization (IVF). C57BL/6 or FVB/N eggs will be inseminated with frozen or fresh sperm (pending import approval by the BIDMC Animal Research Facility). If fresh sperm is used, we will train investigators to cut out the tail of the epididymus. The resulting embryos will be transferred into pseudopregnant recipients. Charge Back Fee = $660.
Consultation. Consultation on all aspects of transgenic and gene knockout work is provided.
* Cost does not include per diems, shipping, etc.
** Albino C57BL/6 embryos facilitate visual detection of chimerism and germline transmission when black C57BL/6 ES clones are being injected. Albino embryos are derived from C57BL/6J-Tyrc-2J mice.
Updated September 18, 2020
After consulting with the Core Director, please use the link below to request Core services.