Hi, I am trying to tag a protein endogenously in Human iPSC model. While reading the protocols, I found different plasmids and I got confused which one works best for hiPCSs.
Can anyone help me and share their experience?
Hi Mamta
there is no preference in hiPSC for a specific plasmid transfection. I personally used several different ones and all worked pretty much fine. more important factor is actually presence of proper selection marker, like resistence to puromycin, or expression of GFP based upon which you can select only the cells which have been succesfully transfected (either by addition of the selection antibiotic, or sort based on presence of the fluorescent marker) so you expand only those that have a potential for DNA cleavage and thus following introduction of your HDR template. so of course it depends if you have the possibility to sort your cells, ideally on plates for clonal expansion. if not, go for antibiotic resistance, but test your cells ahead that they are not already resistant, as that would result in overgrowth of non-transfected cells. then you may proceed to low dilution plating for individual clones growth.
as for promoters, CMV and/or U6 promoters usually present in these plasmids are fine for hiPSC, they are strongly expressed.
Hi Šárka Jelínková
Thanks a lot for the answer. It made prettyy clear to me. Do you also use another plasmid for HDR template or what method do you use to deisgn the HDR template?
Do you have any simplified protocol for CRISPR knock in and tagging of specific gene? would you mind sharing it?
Hi
I often do Knock out and HDR in human iPS cells. The following vectors are recommended for CRISPR / Cas9 expression. PX330-U6-Chimeric_BB-CBh-hSpCas9 provided by Zhang lab. You can purchase it from addgene. Since pX330 is not highly expressed in human iPS cells, Knock out is easy, but the frequency of homologous recombination is low. I have the impression that the Cas9 expression system by the EF1 promoter is superior.
Please refer to this paper.
https://doi.org/10.1016/j.ymeth.2015.10.015
On the other hand, in recent years,
Cas9 and gRNA can be purchased directly as purified proteins and synthetic RNAs. I purchase from IDT (integrated DNA technology). Although expensive, they are simple and very efficient. It's a lot cheaper than making mistakes. Numerous detailed protocols are provided.
The HDR vector has homologous recombination sequences (about 1 kb) corresponding to the cut site(s) on both ends, and drug resistance markers and fluorescent markers are placed between them. An EF1 promoter or CAG promoter should be used to express the marker protein. The CMV promoter will easily disappear.
These are examples of introduction.
gRNA expression pX330 vector: 2µg
Circular fluorescent protein expressing-HDR vector: 8 µg
HiPS cells (Feeder-free cultured): 3x10 ^ 6 cells
NEPA21 electroporater was used for gene-introduction.
It is better to use the HDR vector as it is in circular preventing random integration of the HDR vector. Fluorescence expression will be observed from the next day. Transient fluorescence expression lasts for several days. It is possible to acquire DNA 48 hours after gene transfer and evaluate whether homologous recombination has occurred by PCR analysis. Fluorescence-positive cells are obtained by FACS one week later in order to obtain cells with homologous recombination.
If you reply to this answer, you can have a more detailed discussion.
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