UPDATE 09/18/2013

iPSCs

Mouse iPSCs

Generating transgenic Oct4-GFP mouse iPSCs

Among twenty thousands of genes which mammal expressed, Oct4 is one of the most ancient and essential transcription factors for embryonic stem cells maintenance and embryo development. Oct4 has been longwise recognized as a gatekeeper and marker for pluripotency.

Transgenic Oct4-GFP mice were harbored with an IRES-GFP fusion cassette downstream of the stop codon of endogenous Oct4 gene. When infected with retrovirus which expressed specific transcription factors(Oct4,Sox2,Klf4,cMyc), some Oct4-GFP murine embryonic fibroblasts(MEFs) have the properties of induced pluripotent stem cells(iPSCs). These Oct4-GFP iPSCs whose endogenous Oct4 have been activated will trigger the expression of GFP(enhanced green fluorescence protein) thus to be very convenient to iPSCs verification as well as FCM(flow cytometer) analysis.

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We first breeded transgenic Oct4-GFP C57 BL/6 homozygote F0 male with wild type C57 BL/6 F0 female, and isolated and generated Oct4-GFP primary MEF cell lines from 13.5dpc embryo(Figure 1A). Genome typing was performed to determining F0 homozygote and F1 heterzygote(Figure 1B). We later packaged retrovirus pMXs-Oct4/Sox2/cMyc/Klf4(Constructed by Shinya Yamanaka) in HEK293T. Oct4-GFP MEF cell line, C57-4-A P2, was infected ultracentrifuge enriched viral cocktail. We Replated reprogramming MEFs on mitomycin C treated pMEF-NL feeder layer two days later, and supplied with mouse iPSCs medium from this day on(Figure 1C). iPSCs monoclone formed two weeks after infection(Figure 2)，and this cell lines refer as C57-4-A iPS later.

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Verification of mouse iPSCs:

As C57-4-A iPS was reprogrammed from Oct4-GFP MEFs, we can observe robust GFP expression under inverted fluorescence microscope. Immuno-Fluorescence was also performed to detect some classical pluripotency marker such as Oct4/Nanog(nuclear colocalized) and SSEA1(cytomembrane colocalized).Karyotype analysis demonstrated 53.3% C57-4-A iPS possessing correct chromosome complement(Figure 3).

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Three million C57-4-A iPS cells were injected to three 8 weeks BALBc nu/nu(Nude mice) left shoulder subcutaneously, and same amount of C57-4-A MEFs to the right as control. Four weeks later, significant tumors formed under left(but not the right) shoulder skin of each BALBc nu/nu mouse. Tumors were fixated in 10% formalin, dehydrated, paraffin embedded and HE(hematoxylin and eosin)stainned at last. Various type of tissue developing from entoderm, mesoderm and ectoderm (Figure 4) demonstrated our C57-4-A iPS cell lines not only expressed dogma iPS marker but also can form teratoma in vivo which combined together proved it possessing qualified pluripotency and stemness.

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Stable mouse iPSC line assay:

We infected the lentivirus into iPSCs just three days before the wiki freezing. Because of the time limited, we have to finish the stable test within the number of a 12-well plate of iPSCs. Among them, 7 wells were injected with tTA and Ptight-suicide gene, 4 were added Dox (2000X), one was injected with nothing, 4 are injected with TRE3G-suicide/GFP. The rate of tTA lentivirus and Ptight-suicide gene lentivirus was 1:2, which was the best rate having been proved by the transient experiments we did in Bosc before.

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Without changing the culture solution, after 72 hours, we used the fluorescence-activated cell analyzing for every well of iPSCs. Here are the outputs.

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Figure1.From top to bottom, the test groups are: neg ctrl, TRE3G-GFP, TRE3G-rip1, TRE3G-RIP3, mock+tTA, mock+tTA+Dox, pTight-RIP1+tTA, pTight-RIP1+tTA+Dox, pTight-RIP3+tTA, pTight-RIP3 +tTA+Dox, pTight-VP3+tTA, and pTight-VP3 +tTA+Dox. From left to right, A group represents the density of iPSCs’ GFP(X) and cell size(Y); B group represents the density of iPSCs’ GFP(X) and whole cell numbers(Y); C group represents the density of iPSCs’ GFP(X) and cell size in another tunnel(Y).