Gene Targeting and Stem Cell Facility
Generation of ES Cells from Mouse Embryos
The Gene Targeting/Stem Cell Facility of the TAMC will generate specific ES cell lines from blastocysts isolated from genetically altered mice. Embryos (E3.5) will be plated onto inactivated feeder layers of cells in appropriate media to allow for inner cell mass expansion and embryo hatching. Once the ICM has expanded, the ES cells will be picked and subcloned in a 96-well feeder plate, and subsequently expanded to 24 well plates. DNA will be made from representative clones and provided to the Investigator for testing, and individual ES cell lines cryopreserved and provided to the Investigator. For in-house orders, please contact the TAMC. Third parties, please contact TAMC for further information and to arrange ordering.
Gene Targeting in Mouse ES Cells
The Gene Targeting/Stem Cell Facility of the TAMC will assist in the design of replacement or insertion vectors for gene targeting in ES cells. The Facility has a variety of plasmids available for use in constructing targeting vectors. Upon construction and purification of the targeting vector by the investigator, the Facility will electroporate the vector into mouse embryonic stem cells. Utilizing the appropriate drug selection, the Facility will isolate and array between 300-400 individual ES cell clones in a 96-well format, and will isolate genomic DNA from the clones. Following identification of the correctly targeted clone(s) by the investigator via PCR or Southern analysis, the Facility will expand the clone(s) for further analysis and/or for use in generating chimeric mice. For a description of the time involved for gene targeting, please see timetable. Additional information regarding ES cell lines used by the TAMC can be found here. For in-house orders, please see service charges and service forms. Third parties, please contact TAMC for further information and to arrange ordering.
Nuclease Testing in Mouse ES Cells
The Gene Targeting & Stem Cell Facility can test the efficiency of TALENs or CRISPRs to modify the locus of interest in mammalian diploid cells by electroporating C57BL/6 or 129Sv strain mouse ES cells with 20ug of each TALEN pair (10ug of each half-TALEN plasmid) or with 10-20ug of pX330 (including the guideRNA and Cas9 construct), along with 5ug of a puromycin plasmid (the TAMC provides the requisite PGK-Puro vector DNA). ES cells are transiently selected in Puromycin (on Puro-resistent feeders), and individual clones arrayed on a gelatinized 96-well plate. When the wells are confluent, genomic DNA is processed for each clone and the genomic DNA provided to the Investigator in the 96-well format. These DNA plates can be stored at minus 20C until analyzed. Resuspension of the DNA in 20ul TE is suggested, with 2-4ul to be used in a PCR reaction (the resuspended DNA plates can be stored upright at -20C in case a repeat PCR is needed). Screening 96 clones will establish the actual frequency of target site alteration for any TALEN pair or the efficacy of a Cas9 guide RNA. For in-house orders, please see service charges and contact the TAMC when you are ready to begin analysis.
Generation of iPS Cells Using Mouse or Human Primary Cells
The Gene Targeting & Stem Cell Facility of the TAMC can reprogram mouse or human primary cells to generate induced pluripotent stem cells (iPS).
Mouse cells; the TAMC can start with fresh tail biopsies or frozen or fresh embryonic fibroblasts provided by the Investigator. These cells will be expanded to appropriate densities, and infected with packaged lentivirus bearing the 4 “Yamanaka Factors” (Sox2, Klf4, cMyc, and Oct4) in a Cre-excisable format. Up to 12 colonies displaying undifferentiated morphology will be picked and arrayed on an inactive feeder layer, and expanded to the 6-well stage. Cells will be analyzed for initial pluripotency by alkaline phosphatase activity, and the 6 most primitive clones expanded and confirmed by testing for endogenous pluripotency markers. If viral excision is desired, up to 3 selected clones will be exposed to Cre, subcloned, expanded, and tested by PCR for excision of the lentiviral vector.
Human cells; human primary cells (fibroblasts, myoblasts, or blood cells) at appropriate density will be infected with non-integrating Sendai viral vectors bearing the four “Yamanaka factors”. Up to 12 colonies displaying the most ES cell-like morphology will be picked and expanded onto feeder-cell plates or under the feeder-free, xeno-free conditions. Colonies will be assayed for initial pluripotency by TRA-1 immunostaining. Based upon morphology and AP staining, the 4 “most primitive” clones will be propagated by single colony sub-cloning to facilitate Sendai loss. Immunostaining will be performed to confirm pluripotency and loss of virus.
iPS clones will be provided to the Investigator either as frozen samples or (if pre-arranged) as growing cells. In addition, a flash-frozen aliquot of each clone can be made available for subsequent DNA and RNA analysis. In addition, cells can also be pre-arranged for testing in teratoma formation assays performed by the Animal Modeling Facility. The time-lines and costs for iPS services will vary depending upon the starting material provided to the TAMC and the levels of services desired. For in-house orders, please see service charges and service forms. Third parties, please contact TAMC for further information and to arrange ordering.
Teratoma Formation in Mice to Evaluate Stem Cell Pluripotency
The Gene Targeting/Stem Cell Facility will work with the Animal Modeling Facility of the TAMC to test the capability of iPS cells to form each of the three germ cell layers. Individual iPS clones will be expanded, and injected subcutaneously into the flank of 3 immune-compromised NSG mouse (2 sites/mouse). All iPS cells not generated by the TAMC must be MAP tested (RADIL-2 panel) prior to use in animals. This testing charge is mandated by UMMS IACUC and, if necessary, is recovered from the Investigator. Teratomas will be excised when the in vivo mass reaches 1-2cm in diameter (or if animals exhibit poor health). A portion of each teratoma will be flash-frozen in liquid nitrogen, and the remainder fixed in formalin. For in-house orders, please see service charges and service forms. Third parties, please contact TAMC for further information and to arrange ordering. The TAMC can arrange for histology and pathological analysis of germ cell layer formation for an additional charge.
The TAMC is willing and able to assist Investigators with various procedures not listed above. The Core is also available to consult with investigators regarding their mouse experiments and provide limited training of UMMS researchers, as long as these services do not interfere with the ability of the Core to fulfill its primary mission of creating genetically modified animals and stem cells for the UMASS community in a timely and efficient manner.