iPSC Reprogramming
There are four general groups to categorize an iPSC reprogramming strategy:
Integrative Reprogramming
Integrative Reprogramming techniques require the reprogramming factors to be inserted permanently into the host cell genome. This strategy can be further divided into viral and non-viral methods. The pioneering iPSC experiments by Yamanaka et al., were conducted by integrating four transgenes using retroviral vectors.
ViralThough they are the most efficient method for reprogramming iPSCs, lenti- and retroviral based transduction methods have distinct drawbacks for clinical and translational applications:
Some of these challenges can be addressed with newer technologies. For example, instead of using four separate transgenes with four separate integration sites and, therefore, four chances for mutation, researchers have developed polycistronic lentiviruses, which express all four transgenes sequentially with 2A Peptide self-cleavage sites. Additionally, researchers can flank their transgene insert with loxP sites and use transient transfection with Cre-recombinase to excise the integrated transgenes after reprogramming is complete. |
Schematic of polycistronic insert with Yamanaka factors for iPSC reprogramming. (Top) 2A peptide cleavage sites are highlighted. (Bottom) One solution for removing integrated reprogramming genes is flanking loxP sites. Schematic of loxP sites flanking polycistronic insert.
Non-viral
Transposon technology is a next-generation solution to avoid traditional downfalls of virus transductions. Transposable elements, or transposons, are mobile genomic elements that can move around the genome. In the case of genetic editing and iPSC generation, it refers to a plasmid carrying a transposon with all the reprogramming elements.
TcBusterTM is a transposon-based technology developed by Bio-Techne. In addition to reducing mutagenic potential and cargo-size constraints conferred by traditional viral transduction, gene editing with TcBuster is faster and safer for clinical and translational samples. TcBuster technology can also be used to engineer additional genetic changes in iPSCs.
Non-integrative ReprogrammingNon-integrative Reprogramming techniques are the preferred methodology for clinical and translational iPSC generation. They require no genomic integration, and therefore have significantly reduced chance of introducing harmful mutations. However, non-integrative methods are less efficient, and expression of reprogramming factors is more transient. If non-integrative methods are chosen, there are many small molecules available to increase reprogramming success.
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