We performed six patterns of PCR in parallel using 2 ng DNA for each reaction. In the first step of PCR, we performed 40-cycle PCR to amplify vector-genome junctions using PrimeSTAR GXL DNA Polymerase (TaKaRa Bio, Ohtsu, Japan), a Mastercycler pro S (Eppendorf, Hamburg, Germany), and primers, which were specific to an end of the transgene and adapter sequence, according to the manufacturers' instructions. Tag-PCR mapping is a useful technique for assessing the risk of insertional mutagenesis. Integration into or near proto-oncogenes was similar in all three systems. The piggyBac system demonstrated lower preference than the retroviral system for integration near transcriptional start sites and CpG islands and higher preference than the lentiviral system for integration into genomic safe harbors. Using this system, we compared the integration sites of CD19-specific CAR genes in final T cell products generated by retrovirus-based and lentivirus-based gene transfer and by the piggyBac transposon system. ![]() Here we describe a new tagmentation-assisted PCR (tag-PCR) system that can determine the integration sites of transgenes without using restriction enzyme digestion (which can potentially bias the detection) and allows library preparation in fewer steps than with other methods. Insertional mutagenesis is an important risk with all genetically modified cell therapies, including chimeric antigen receptor (CAR)-T cell therapy used for hematological malignancies. The Lancet Regional Health – Western Pacific.The Lancet Regional Health – Southeast Asia.The Lancet Gastroenterology & Hepatology.
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