Year: 2019
Type: preprint
Abstract: While transgenic populations may be enriched to near-purity using the techniques described above, the derivation of a clonal iPSC line descended from a single parent cell is necessary for proper genotyping and for many downstream applications, and it is thus standard practice following any genetic edit. One common method for isolating such clones, as described below, is by serial dilutions on two 6-well plates, followed by manual picking. As with all single-cell dissociations of iPSCs, the use of Y-27632 ROCK inhibitor is required until colonies are properly established; wells with fewer cells may require treatment for 2 to 3 days. 6-well plates are used because their wells have a larger surface area than other multiwell dishes, facilitating downstream picking. For highly enriched populations, serial dilutions may be reduced to one 6-well plate loaded with 1 × 105 cells in the first well, as there is a higher probability of identifying a purely positive colony. Following isolation, this protocol further describes basic genotyping in parallel with the gradual expansion of clonal lines, utilizing QuickExtract to prepare genomic DNA and PCR to test for the presence of a transgene of interest. Primer sequences and other specifics are included in more detail for the particular differentiation cassettes in Support Protocol 1. In general, basic genotyping by PCR should be performed as quickly as possible to screen out negative clones, while potentially positive clones should be confirmed by more stringent methods such as Sanger sequencing and western blotting as appropriate. To save culture reagents, negative clones may be immediately discarded, while all others should be expanded and frozen pending confirmation.
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
OpenAlex ID: W3012054138