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We use high quality human foreskin fibroblasts for reprogramming. These can be obtained through a few sources, however, Life Line Cell Technology has xeno-free system (robust cells and matching media) which are both consistent in our hands. More infomation can be found at this link.
If you have your own sources, consider trying their media as your cells will grow more rapidly, and reprogram even into passage 7 (latest passage we've tried).
We use 4 genes in a few different self-inactivating lentiviral formats to reprogram cells into IPSCs. We are now developing a simple non-genetically modifying format! More information to follow shortly.
These are Oct4, Sox2, KLF4 and L-Myc. No more C-Myc!
We have a 1, a 2 and 4 virus system to do the job.
The 1 virus system is comprised of a polycistronic super-gene with fluoscent detection and antibody selectability. The two virus system has no Myc component, this allows to search for a substitute that permit efficient reprogramming. The four virus system has Oct4-IRES-RFP/Blast, and other single factors without color but with Puro selection.
Please note that optimized starting material (we use human foreskin fibroblasts) and media schema are equally important as viral tools for successful IPSC generation. If you need help with protocols, we can suggest our refinements which have produced presumptive IPSCs colony formation within 10 days!
Our systems are available upon request.
a) 1 Virus system - Oct4-2A-Sox2-2A-KLF4-2A-L-Myc-2A-RFP/Blast fusion.
b) 2 Virus system - Oct4-2A-Sox2-2A-KLF4-2A-RFP/Blast
with choice of...
i) c-Myc (rapid, many false positives)
ii) L-Myc (intermediate kinetics, but almost only properly formed colonies)
iii) Glis1 (slower, very small granular, longer kinetics with some propely formed colonies)
c) 4 Virus system with Oct4-IRES-RFP/Puro, Sox2, KLF4, and L-Myc (c-Myc, or Glis1).
Sample iPSC colony with self-forming feeder layers.
Please note, there is no financial gain for the above mentioned endorsements. These photographs are property of Rutgers University, and Rick Cohen, Ph.D., and may not be used without expressed written permission.