| Keywords [eng] |
iPSC, induced pluripotent stem cells, indukuotos pluripotentinės kamieninės ląstelės, Jurkat, Cas9, genome editing, genomo redagavimas, polyengineering, poliinžinerija |
| Abstract [eng] |
Genomic editing of cell lines is essential in developmental biology, disease modelling and clinical research. However, usually, only one or a few genes are edited in the same cell line, limiting the capability of research into gene interactions. A strategy for editing many loci in the same cell line could be beneficial for research on interactions of genes, providing a method for the detailed study of both healthy and disordered development. This project focuses on the design of a strategy for polyengineering knockouts by HDR-based genome editing. The aim of the research was to create plasmid constructs for the knockouts of the genes SOX1, PAX6, EOMES, GSC, FOXG1, OTX2, SOX17, EN1, HOXA2, HOXB4 and establish a human Cas9 knock-in cell line, using HDR-based genome editing. Plasmids with a positive selection model were created by In-Fusion cloning for the knockout of genes SOX1, PAX6, EOMES and SOX17, all vital for differentiation. A Jurkat cell line with Cas9 knock-in was created for stable expression of Cas9, useful in editing multiple loci of the same cell line. The success of the knock-in was verified by genotyping PCR. |
