Generation of Human Induced Pluripotent Stem Cell Line Carrying TIA1 Gene Mutation by CRISPR/Cas9 System

Using CRISPR/Cas9-mediated genome editing, a human iPSCs (induced pluripotent stem cells) line carrying TIA1-P362L mutation was generated and differentiated into motor neurons, providing a useful cell model for studying the molecular mechanisms of ALS (amyotrophic lateral sclerosis) and drug screening. sgRNA was designed using the CRISPR online design tool, and was ligated into the pX330 vector as the targeting plasmid. Meanwhile, left and right arms for homologous recombination were amplified and inserted into a Donor vector, which was mutated subsequently at target site to construct the Donor plasmid for mutation. The two plasmids were co-transfected into the iPSCs line C11. After screened for G418 resistance, single clones were selected and expanded. PCR and Sanger sequencing were applied to validate the genotype of the selected clones. The expression of TIA1 was identified by Western blot. Pluripotency of the TIA1-P362L iPSCs line was analyzed by karyotyping, immunofluorescence, quantitative reverse transcription PCR and three-germ-layer differentiation of EBs (embryoid bodies). The TIA1- P362L iPSCs were further differentiated into motor neurons by inhibition of SMAD pathway and immunofluorescence was used to validate the expression of specific motor neuron markers. The above study demonstrated that an iPSCs line carrying TIA1-P362L mutation was successfully constructed, which had normal pluripotency capacity and could be differentiated into motor neurons, providing a resource for elucidating the role of TIA1 in ALS disease.

CSTR: 32200.14.cjcb.2023.09.0007