How iPSCs are made?

We apply the integrative Lenti-virus delivery (LV) system, or non-integrative systems, such as Sendai-virus delivery (SV) and episomal vectors (EV). 

We are happy to assist you, choosing the right reprogramming system suited for your application need. 

Lenti-virus delivery (integrated transgene) 

We use a Polycistronic LV-delivery system, containing the OCT4, KLF4, SOX2, cMYC transcription factor (TF) and dTomato marker sequences [18;19]. The transgene stably integrates into the host genome and its self-silencing property assures proper differentiation capability of the generated iPSC lines. 

Sendai-virus delivery (transgene-free)

One of the transgene-free system based on SV-delivery, where we use the commercially available (ThermoFisher) SV-reprogramming viruses [17]. Sendai-virus is an RNA based virus, remains in the cytoplasm, thus eliminating integration into the host genome. Upon reprogramming the SV backbone and transgenes clear from the cytoplasm, thus the iPSC lines become transgene-free. To confirm the transgene-free status, SV-specific RT-PCR is used. 

Episomal plasmid (transgene-free)

The other transgene-free reprogramming system we routinely apply is based on episomal plasmids which are delivered into the host cells by nucleofection [15; 16]. Upon delivery, the episomal plasmids containing the pluripotency TFs are expressing from the cytoplasm without integration into the host genome. Due to the rapid replication rate of the host cells, the episomal vectors dilutes out of the system. We confirm the transgene-free status of the iPSCs by EV-specific gPCR. 

Contact

Questions or want to order?

ipsc.facility@sanquin.nl