Responsibilities of the Investigator

• Provide the facility with a vial of frozen cells or a plate of growing ES cells.
• Provide a protocol for culture.
• Submit a Request Form to initiate your project.

Services Provided by the Facility

• The facility will culture your ES cells in the appropriate medium, expand and freeze several aliquots.
• Frozen cells will be maintained in the facility's auto-fill liquid nitrogen storage tank, unless requested otherwise by the investigator.

The Transgenic Facility is now providing mouse embryonic fibroblasts/feeders, both active and post-mitotic. Fibroblasts are from the DR4 mouse line, with resistance to neomycin, puromycin, hygromycin and 6-thioguanine.  Upon receiving a request, the Facility will plate the inactivated feeders in the format you choose: 6-well, 12-well, 24-well, 96-well, or 10 cm dish.  

*Please provide your request several days in advance of requiring delivery of the feeder plate(s).*  

Cost is $75 per plate, regardless of format.

Responsibilities of the Investigator

• Discuss experimental design with facility directors before beginning your project.
• Provide a map of the construct including size, regions of homology, selection strategy, enzyme sites and oligo binding sites.
• Identify a unique restriction site to linearize your plasmid.  Provide gel documentation of your test digest.
• Provide 100ug of purified plasmid at 1ug/ul.
• Develop a reliable genotyping assay (long PCR, Southern, or both) before submission of DNA to the facility.
• Screen picked clones for targeted candidates within three months (during which time the clones are stored at -80^o).
• Confirm genotype of candidate clones by Southern blot.
• Submit a Request Form in iLabs to initiate the project.

Services Provided by the Facility

• The gene-targeting facility will linearize your plasmid, electroporate into ES cells, perform selection and pick clones.
• ES cell lines provided:
  • E14 (129/Ola parental ESC line)
  • EDJ22 (129/SvEv parental ESC line)
  • JM8.N4 (C57Bl/6N parental ESC line)
  • JM8.A3 (C57Bl/6N agouti-rescued parental ESC line)
• We guarantee two rounds of electroporations (if necessary) for each targeting construct.
• Picked clones will be stored at -80^o for up to three months, during which time the investigator must screen for candidate clones.
• The facility will expand candidate clones for freezing and confirmation of genotyping by Southern blot (investigator provides Southern blot confirmation).
• After confirmation of homologous recombination, the facility will karyotype up to four of these clones.  Any additional expansion and/or karyotyping of clones will be subject to an additional charge.

Responsibilities of the Investigator

• Provide the facility with a frozen vial of ES/iPS cells or a plate of growing cells.
• Provide a protocol for culture of your ES/iPS cells.
• Submit a Request Form in iLabs to initiate your project.

Services Provided by the Facility

The Pluripotent Stem Cell Core of the Transgenic and Gene Targeting Facility conducts a simple chromosome counting procedure to determine the percentage of euploid cells.  No advanced karyotypic analysis is offered.

• The facility will culture and arrest pluripotent stem cells in metaphase with colcemid.
• We will prepare and count 20 chromosome spreads following a basic Giesma staining protocol.
• The percentage of euploid cells will be provided to the investigator.

The Transgenic and Gene Targeting Facility can derive primary human fibroblasts from biopsy/autopsy tissues.

Responsibilities of the Investigator

• Provide the facility with living tissue in DMEM/RPMI at room temperature or on ice.
• Submit a Request Form in iLabs to initiate your project.

Services Provided by the Facility

• The facility will derive primary fibroblasts from the tissue in the appropriate medium and freeze several aliquots.
• Frozen cells will be maintained in the facility's auto-fill liquid nitrogen storage tank, unless requested otherwise by the investigator.

The Transgenic and Gene Targeting Institutional Facility can reprogram primary fibroblasts into iPS cells using the Sendai Virus. Contact Jay Vivian for more information. 

Responsibilities of the Investigator

• Provide the facility with a vial of frozen cells or a plate of growing ES/iPS cells.
• Provide a protocol for culture.
• Submit a Request Form in iLabs to initiate your project.

Services Provided by the Facility

• The facility will culture your ES/iPS cells in the appropriate medium, expand and freeze several aliquots.
• Frozen cells will be maintained in the facility's auto-fill liquid nitrogen storage tank, unless requested otherwise by the investigator.

Responsibilities of the Investigator

• Discuss experimental design with facility directors before beginning your project.
• Provide a map of the construct including size, regions of homology, selection strategy, enzyme sites and oligo binding sites.
• Identify a unique restriction site to linearize your plasmid.  Provide gel documentation of your test digest.
• Provide 100ug of purified plasmid at 1ug/ul.
• Develop a reliable genotyping assay (long PCR, Southern, or both) before submission of DNA to the facility.
• Screen picked clones for targeted candidates within one month (during which time the clones are stored at -80^o).
• Confirm genotype of candidate clones by Southern blot.
• Submit a Request Form in iLabs to initiate the project.

Services Provided by the Facility

• The gene-targeting facility will linearize your plasmid, electroporate into ES/iPS cells, perform selection and pick clones.
• ES cell lines provided:
  • H9 (WA09)
  • H1 (WA01)
• iPS cell lines provided: 
  • DF6-9-9T.B
  • 11b (feeder-dependent)
• We guarantee two rounds of electroporations (if necessary) for each targeting construct.
• Picked clones will be stored at -80^o for up to one month, during which time the investigator must screen for candidate clones.
• The facility will expand candidate clones for freezing and confirmation of genotyping by Southern blot (investigator provides Southern blot confirmation).
• After confirmation of homologous recombination, the facility will karyotype up to four of these clones.  Any additional expansion and/or karyotyping of clones will be subject to an additional charge.

The Transgenic and Gene Targeting Institutional Facility can differentiate hES/iPS cells via embryoid bodies. Contact Jay Vivian for more information. 

ES Cell Lines Provided: 

• H9 (WA09)
• H1 (WA01)

iPS Cell Lines Provided: 

• DF6-9-9T.B
• 11b (feeder-dependent)

The Transgenic and Gene Targeting Institutional Facility can assess the capacity of a cell line to differentiate into three germ layers in vivo via the teratoma assay.

Responsibilities of the Investigator

• Provide the facility with a vial of frozen cells or a plate of growing cells.
• Provide a protocol for culture.
• Submit a Request Form in iLabs to initiate your project.

Services Provided by the Facility

• The facility will culture your cells in the appropriate medium and inject them into mice testes.
• The facility will conduct histological and pathological analysis of the teratoma.  

The Facility can perform a PCR assay to test for the presence of over 60 species of mycoplasma in cell culture. 

Investigator should provide the facility with cells in a 1.5 ml tube: 

• Suspension cells: count 10⁴ - 10⁵ cells and resuspend in 1 ml PBS or media 
• Adherent cells: scrape cells from one well of a 6-well plate and resuspend in 1 ml of PBS or media
  •  Do not dissociate cells with trypsin or EDTA; these agents disrupt mycoplasma. 

Cell suspension should be frozen and stored at -80. 

The facility can perform CRISRP-based gene targeting in a variety of cell lines. Contact Jay Vivian for more information.