Human Pluripotent Stem Cell Gene Targeting

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.