Upon receiving your lab’s DNA construct, the Gene Targeting and Transgenic Shared Resource will perform an electroporation of your DNA into W4 (129S6/SVEvTac), G4 (129/B6) or JM8A3.N1 (C57BL/6Tac) ES cells. Colonies will be selected with the appropriate drug, depending on your positive selectable marker. Colonies can also be negatively selected for enrichment purposes. Selection methods should be discussed with the resource prior to the electroporation date to prepare the necessary feeder cells.
The resource will pick and freeze ~240 colonies, of which we will give the investigator’s lab ~200 DNA preps. The investigator will screen these clones by Southern analysis, and/or PCR, and notify the resource of the positives for homologous recombination. The facility will expand five of these clones and give the investigator DNA again to reconfirm positives. When reconfirmed, the resource can begin Gene Targeting microinjections for the investigator's laboratory.
The entire electroporation process takes approximately one month to complete and the resource expects the investigator’s lab to perform the positive confirmations in a timely fashion.
Please contact Aimee Stablewski at 716-845-5843 or Aimee.Stablewski@roswellpark.org for a project specific cost estimate.