I want to do hESC differentiation with some TFs, which work at different stages. The ef1a promoter drive genes without control. Dox-TRE system can only control one stage? How to control different TF expression in several stages? For example, stage I express A and B, stage II express C and D, stage III express E and F. As I know oct4 promoter is huge, does it ok for Lentiviral Vectors?
Hey,
I dont get your pint completely. You want to have a time point specific expression of some of your genes? I guess it will be hard to get this done, because in case you use inducible promotors you would have to also transduce the corresponding repressor plamsids. This seems not so applicable. The size of your insert withing a lentiviral construct could go up to 6-8 kb but should not exceed his values. 1 Kb insert size is reducing your transduction efficiency ten-fold, means a 2 kb construct is 20 times less efficient compared to the corresponding empty vector. It works with big inserts but you need more virus probably
Regards Sven
What about transfecting your clls with mRNA or recombinant proteins instead of plasmids? This would give you the chance to do precise timed expression settings
Hello Hyde,
Differential inducible system seems a complicated strategy.
However you could use a set of non integrating lentiviral vectors (NILV) with differential transductions :
Stage 1 :transduction with NILV A and B, stage 2 transduction with NILV C and D ......
Best,
Nicolas
@ Nicolas,
Do you think cells can be transduces as often as needed consecutively? This is real curiousity, because I woud assume that 3 different particles would already be a lot but my experience with lentivirus did not go that far
Regards Sven
@Sven,
I already did this type of strategy, I transduced the cells with NILV every three days during 4 weeks without any problems
Nicolas
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