Director:Bradford B. Lowell, MD, PhD Professor of Medicine, BIDMC and Harvard Med. School
Co-Director:Dong Kong, PhD Asst. Professor of Neuroscience, Tufts University School of Medicine
Co-Director:Chen Wu, PhD Instructor, BIDMC & Harvard Medical School
BIDMC Facility Director:Joel Lawitts, PhD Instructor, BIDMC and Harvard Med. School
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)
- 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 = $2,500 for FVB transgenics and $3,850 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 $600* for FVB/N and $1,200 * for C57Bl/6.
Guarantee: 4 founders. If more than 50 pups were born without achieving 4 founders, injection can be repeated with above-mentioned DNA re-prep fee.
- 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 = $2,500 for FVB transgenics and $3,850 for C57BL/6 transgenics. Consultation and questions on all aspects of using CRISPR or Easi-CRISPR to make genetically modified mice is provide by Chen Wu (firstname.lastname@example.org) and Brad Lowell (email@example.com).
If repeat injections are required, no new reagents will be accepted. A re-injection fee of $600* for FVB/N and $1,200* for C57Bl/6 will be applied. Submission of new reagents will be billed as 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 = $1,750.
- 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 = $750. Charge Back Fee for 1 day’s injection (either 1 or 2 ES clones) into albino** C57BL/6 embryos = $900.
- Cryopreservation of Mutant Mice. Fertilized one cell embryos are cryopreserved. Charge Back Fee = $3.50 per mouse embryo.
- 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 = $550.
- In Vitro Fertilization (IVF). B6D2F1 hybrid 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 = $450.
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.
After consulting with the Core Director, please use the link below to request Core services.