Transgenic Mouse Core

The Transgenic Mouse Core was established in 1992 operating at the BWH, and is now located at Harvard Medical School. The Transgenic Mouse Core provides services for the generation of transgenic and knockout mice using state-of-the-art facilities and equipment.

The Core has all of the equipment necessary for generation of transgenic mice including a Nikon Diaphot microscope equipped with Nomarski Optics and Narishige micromanipulators for microinjections, Nikon surgical microscopes for egg isolation and transfer, a sutter needle puller and a de Fonbrune microforge. The facility maintains mice necessary for egg donors, egg recipients, and vasectomized males.

ES cell culture services have been offered for 15 years. The core offers electroporation of targeting vectors into ES cells and provides investigators with DNA to identify ES cells carrying the desired recombination events; and ES cell expansion for microinjection into blastocysts.

The core's newest services include CRISPR injections, sperm and embryo cryopreservation, and reanimation with fresh or frozen sperm via IVF or the thawing and implanting embryos.

Key Services

  • CRISPR Injection services
  • ES cell electroporation services
  • ES cell injection services
  • Fresh/Frozen sperm IVF
  • Inducible progenitor stem cells (IPS) injection
  • Injection of DNA constructs (BALB C, C57BL/6, FVB) into egg pronuclei for generating transgenic mice
  • Lentiviral Injection services
  • Sperm/embryo cryopreservation
  • Tail biopsy services
  • Thawing and implantation of embryos

For more information on services go to the Harvard medical Area Core Management System at: pathcore.hms.harvard.edu.

Highlighted Projects

An obligate cell-intrinsic function for CD28 in Tregs.

PI:  L. TurkaBIDMC, MGH (Cancer Immunology Program)

Tregs expressing the transcription factor FOXP3 are critical for immune homeostasis. The costimulatory molecule CD28 is required for optimal activation and function of naive T cells; however, its role in Treg function has been difficult to dissect, as CD28 is required for thymic Treg development, and blockade of CD28-ligand interactions has confounding effects in trans on nonregulatory cells. To address this question, we created Treg-specific Cd28 conditional knockout mice. Despite the presence of normal numbers of FOXP3+ cells, these animals accumulated large numbers of activated T cells, developed severe autoimmunity that primarily affected the skin and lungs, and failed to appropriately resolve induced experimental allergic encephalomyelitis. This in vivo functional impairment was accompanied by dampened expression of CTLA-4, PD-1, and CCR6. Disease occurrence was not due to subversion of Cd28-deficient Tregs into pathogenic cells, as complementation with normal Tregs prevented disease occurrence. Interestingly, in these "competitive" environments, Cd28-deficient Tregs exhibited a pronounced proliferative/survival disadvantage. These data demonstrate clear postmaturational roles for CD28 in FOXP3+ Tregs and provide mechanisms which we believe to be novel to explain how interruption of CD28-ligand interactions may enhance immune responses independent of effects on thymic development or on other cell types.

Contribution of the Core: The Core carried out the embryo stem cell work on the C57BL/6 background to generate the Cd28-ΔTreg mice. 

Publication:  Zhang R, Huynh A, Whitcher G, Chang J, Maltzman JS, Turka LA.  An obligate cell-intrinsic function for CD28 in Tregs.  J Clin Invest. 2013 Feb;123(2):580-93. PMC3561819.

Deletion of CTLA-4 on regulatory T cells during adulthood leads to resistance to autoimmunity.

PIs: A. SharpeHMS (Cancer Genetics Program, Cancer Immunology Program); V. KuchrooBWH (Cancer Immunology Program); and G. FreemanDFCI (Cancer Immunology Program, Melanoma Program)

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential negative regulator of T cell responses. Germline Ctla4 deficiency is lethal, making investigation of the function of CTLA-4 on mature T cells challenging. To elucidate the function of CTLA-4 on mature T cells, we have conditionally ablated Ctla4 in adult mice. We show that, in contrast to germline knockout mice, deletion of Ctla4 during adulthood does not precipitate systemic autoimmunity, but surprisingly confers protection from experimental autoimmune encephalomyelitis (EAE) and does not lead to increased resistance to MC38 tumors. Deletion of Ctla4 during adulthood was accompanied by activation and expansion of both conventional CD4+Foxp3− (T conv) and regulatory Foxp3+ (T reg cells) T cell subsets; however, deletion of CTLA-4 on T reg cells was necessary and sufficient for protection from EAE. CTLA-4 deleted T reg cells remained functionally suppressive. Deletion of Ctla4 on T reg cells alone or on all adult T cells led to major changes in the Ctla4sufficient T conv cell compartment, including up-regulation of immunoinhibitory molecules IL-10, LAG-3 and PD-1, thereby providing a compensatory immunosuppressive mechanism. Collectively, our findings point to a profound role for CTLA-4 on T reg cells in limiting their peripheral expansion and activation, thereby regulating the phenotype and function of T conv cells.

Contribution of the Core: The core carried out the embryo stem cell work to generate the CTLA-4 conditional knockout mouse Es cells on the C57BL/6 background and performed the blastocyst injection to generate the mice. 

Publication: Paterson AM, Lovitch SB, Sage PT, Juneja VR, Lee Y, Trombley JD, Arancibia-Cárcamo CV, Sobel RA, Rudensky AY, Kuchroo VK, Freeman GJ, Sharpe AH. Deletion of CTLA-4 on regulatory T cells during adulthood leads to resistance to autoimmunity. J Exp Med. 2015 Sep 21;212(10):1603-21.PMC4577848.

Defect in regulatory B-cell function and development of systemic autoimmunity in T-cell Ig mucin 1 (Tim-1) mucin domain-mutant mice.  

PI:  V. KuchrooBWH (Cancer Immunology Program); J. BonventreBWH (Kidney Cancer Program); and S. XiaoBWH (Cancer Genetics)

Tim-1, a type I transmembrane glycoprotein, consists of an IgV domain and a mucin domain. The IgV domain is essential for binding Tim-1 to its ligands, but little is known about the role of the mucin domain, even though genetic association of TIM-1 with atopy/asthma has been linked to the length of mucin domain. We generated a Tim-1–mutant mouse (Tim-1Δmucin) in which the mucin domain was deleted genetically. The mutant mice showed a profound defect in IL-10 production from regulatory B cells (Bregs). Associated with the loss of IL-10 production in B cells, older Tim-1Δmucin mice developed spontaneous autoimmunity associated with hyperactive T cells, with increased production of IFN-γ and elevated serum levels of Ig and autoantibodies. However, Tim-1Δmucin mice did not develop frank systemic autoimmune disease unless they were crossed onto the Fas-mutant lpr mice on a C57BL/6 background. Tim-1Δmucinlpr mice developed accelerated and fulminant systemic autoimmunity with accumulation of abnormal double-negative T cells and autoantibodies to a number of lupus-associated autoantigens. Thus, Tim-1 plays a critical role in maintaining suppressive Breg function, and our data also demonstrate an unexpected role of the Tim-1 mucin domain in regulating Breg function and maintaining self-tolerance.

Contribution of the Core: The Core carried out the embryo stem cell work to generate the Tim-1Δmucin mice on the C57BL/6 background. 

Publication: Xiao S, Brooks CR, Zhu C, Wu C, Sweere JM, Petecka S, Yeste A, Quintana FJ, Ichimura T, Sobel RA, Bonventre JV, Kuchroo VK. Defect in regulatory B-cell function and development of systemic autoimmunity in T-cell Ig mucin 1 (Tim-1) mucin domain-mutant mice. Proc Natl Acad Sci U S A. 2012 Jul 24;109(30):12105-10. PMC3409739.

Publication Acknowledgement:

If research supported by this core facility results in publication, please acknowledge this support by including the following in your publication(s):
"We thank Dana-Farber/Harvard Cancer Center in Boston, MA, for the use of the Transgenic Mouse Core, which provided __________ service. Dana-Farber/Harvard Cancer Center is supported in part by an NCI Cancer Center Support Grant # P30CA006516"