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Team:SYSU-China/Project/result/stable assay
From 2013.igem.org
ipsc
iPSCs
The fate of a unfortunate iPSCs.
Mouse iPSCs
Generating transgenic Oct4-GFP mouse iPSCs
Among dozens of 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(green fluorescence protein) thus to be very convenient to iPSCs verification as well as FCM(flow cytometer) analysis.
Figure 2. Flow chart for generation of mouse iPSCs. Wild type C57 BL/6 female was injected with 5IU PMSG,and 5IU hCG 48h later, then mated with transgenic C57 BL/6 Oct4-GFP(OG) male.OG MEF was isolated and generated from 13.5 dpc embryo back tissue.GFP specific primer was used to perform genome PCR and essured F0 homozygote and F0 heterzygote.Retrovirus pMXs-Oct4/Sox2/Klf4/cMyc(OSKM) was packaged in HEK293T and ultalcentrifuged to enriching viral titer.OG MEF was infected with OSKM viral cocktail and replated on feeder layer 48h later.10 days after viral infection could we observe iPS clone formation.
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.
Figure 3. Morphology during reprogramming.
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).
Figure 4. Pluripotency marker identification. iPSCs GFP expression was detected through FITC filter.Karyotype analysis revealed 2n=40 intact mouse chromosome complement.Immuno-Fluorescence stainning proved Oct4/Nanog/SSEA-1 expression in C57-4-A iPS.
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.
Figure 5. iPSCs_Teratoma HE stainning. teratoma was formed in BALBc nu/nu(nude mice) developing from three million iPSCs. Hematoxylin and eosin(HE) stainning brought out intestines ,lung ,bladder tissue indicated that iPSCs prossess qualified pluripotency.
Stable mouse iPSC line assay:
iPSCs was infected with lentivirus just three days before the wiki freezing. Because the time was limited, we have to finish the stable line 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.
With routine medium refresh, 72 hours later, we ran FACS Calibur flow cytometer to identify the iPSCs(with robust GFP signal) and differentiated cells. Here are the outputs.
Figure6. 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).
From the figure above, what we can observe is as follows:
1. The number of cells infected with both tTA and Ptight-suicide gene (RIP1, RIP3, VIP3) decreases significantly, while that of cells only infected with TRE3G, which has been proven before to have a higher expression than Ptight, is relatively close to the negative control group. And in the survived cells infected with both tTA and Ptight-suicide gene, most cells have differentiated. This result proved that each of these three suicide genes not only have significant killing effects in mouse iPSCs, but also destroy the features of iPSCs, causing them easier to differentiate.
2. The cells infected with TRE3G live quite normally. This indicates that the leaky expression of TRE 3G in stable cell lines is tolerable, which conflicts with the result of transient transfection with TRE3G in Bosc.
3. The cells infected with tTA and Dox live normally. This indicates that Dox has little or no side effect for iPSC features, which eliminates the biggest worry of us before.
4. The cells infected with tTA, Ptight-suicide gene and Dox(2000x) don’t show a rescue effect compared with the cells without Dox. This result indicates that something is wrong with tet-off system, probably in the combination of tTA and TRE, given that the number of cells only infected with tTA and Dox is much more than the cells infected with tTA, Ptight-suicide gene and Dox. Here are the statistic table showing the number and rate of survived cells and survived iPSCs.
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