Issue link: http://life-technologies.uberflip.com/i/215145
Cell line authentication To confirm the identity of the derived iPSC clones in comparison to their parental fibroblasts, genomic DNA was isolated from Parkinson's donor fibroblasts and their subsequent reprogrammed iPSCs. The AuthentiFiler™ PCR Amplification Kit was used to confirm that iPSCs shared the same short tandem repeat (STR) profiles, or "genetic fingerprints", with their parental fibroblasts. The AuthentiFiler™ PCR Amplification Kit is a short tandem repeat multiplex PCR assay that amplifies 9 unique STR loci and the amelogenin gender-determining marker in a single PCR amplification. Briefly, PCR was performed on the Veriti® 96-Well Thermal Cycler, following the recommended cycling conditions for the kit, using the genomic DNA samples and the AuthentiFiler™ Master Mix and Primer Set. Genetic sequencing was then performed on the PCR products via capillary electrophoresis using a 3130xl Genetic Analyzer. After electrophoresis, GeneMapper® ID-X Software was used to genotype and compare the sample profile. A representative result is shown in Table 2 for the MSA and age-matched control (Ctrl-2) lines. Comparative analysis demonstrates identical STR genotypes in the starting fibroblast material and the corresponding iPSCs. This suggests no significant gene duplication or deletion events occurred in the target loci chromosomes, and additionally, no cross-contamination events occurred in which other non-target human cells were introduced into the cell culture. Both of these results provide confidence and verification that the iPSC material is genetically identical to the starting fibroblast material. 24 Life Technologies | Parkinson's cell model Table 2. Cell line authentication. Locus Chromosome MSA donor Age-matched control Fibroblast iPSC Fibroblast iPSC D10S1248 10q26.3 13, 16 13, 16 14, 14 14, 14 D1S1656 1q42.2 16, 17.3 16, 17.3 15, 16.3 15, 16.3 X X, X X, X X, X X, X 2q35 17, 19 17, 19 22, 26 22, 26 22q12.3 16, 16 16, 16 16, 16 16, 16 19q12 14, 14 14, 14 13, 14 13, 14 11p15.5 6, 9.3 6, 9.3 9, 9.3 9, 9.3 2p14 8, 11 8, 11 11, 11 11, 11 D6S1043 6q16.1 12, 12 12, 12 12, 19 12, 19 D12S391 12p13.2 18, 22 18, 22 17, 20 17, 20 AMEL* D2S1338 D22S1045 D19S433 TH01 D2S441 *AMEL (amelogenin) marker "X" indicates female donor.