General Description

Monitor myogenesis in real-time

With SBI’s pGreenZeo, pRedZeo, and pRedTK line of differentiation reporter vectors, you can monitor stem cell differentiation in real-time. These vectors take advantage of our reliable lentivector technology and save you time: our pre-engineered differentiation reporters come as ready-to-package lentivector plasmids or as packaged-to-transduce lentiviruses. Mouse myogenin pGreenZeo differentiation indicator co-expresses dscGFP (destabilized copGFP, 2-hour half-life) and zeomycin resistance of mouse myogenin promoter/enhancer elements, allowing visualization of myogenesis by GFP fluorescence and the selection of the desired cells by zeomycin.

• Create stable cell lines that report myogenesis
• Monitor multiple lineages simultaneously
• Monitoring of differentiation in living cells in real-time
• Note that these vectors only work correctly when transduced. Transfection keeps the constitutive RSV promoter intact, leading to nonspecific expression of reporter genes.

Background

Myogenin is a transcription factor that is expressed during terminal myoblast differentiation in embryonic development and adult muscle regeneration. Investigation of this cell state transition has been hampered by the lack of a sensitive reporter to dynamically track cells during differentiation.

Results

Here, we report a knock-in mouse line that expresses the fluorescent protein tdTOMATE from the endogenous Myogenin locus. Expression of tdTOMATE in MyogntdTom mice recapitulated endogenous myogenin expression during embryonic muscle formation and adult regeneration and allowed isolation of the MYOGENIN + cell population.

We also show that tdTOMATE fluorescence enables the monitoring of differentiation myoblasts in vitro and by intravital imaging in vivo. Finally, we monitor the dynamics of cell division of differentiating myoblasts in vitro using live imaging and show that a fraction of the MIOGENIN + population can undergo a round of cell division, although with a much lower frequency than MIOGENIN myoblasts -.

Conclusions

We hope that this reporter mouse will be a valuable resource for researchers investigating skeletal muscle biology in developmental and adult contexts.

Materials and methods

1. Mouse maintenance

The animals were handled in accordance with national and European Community guidelines and an ethics committee of the Institut Pasteur (CETEA, Comité Ethique en Expérimentation Animale) in France approved the protocols (License 2015-0008). Except where otherwise indicated, 2- to 4-month-old males and females were used.

2. Generation of the Myog-and tomato construct for CRISPR-Cas9-mediated homologous recombination

A 1000 bp fragment of the last exon of Myog was amplified by PCR from murine gDNA (primers 1 and 2, Supplementary Table 1), introducing SalI and NotI restriction sites. This fragment was subcloned into the donor plasmid encoding tdTOM (kind gift of Dr. Festuccia, Institut Pasteur). A 760 bp fragment of the 3’UTR of the Myog gene just after the STOP codon was amplified by PCR from murine gDNA (primers 3 and 4). This amplification also introduced a mutation in the PAM sequence necessary for editing the CRISPR-Cas9 genome.

Using the added PacI and SpeI restriction sites, the fragment was subcloned into the PacI and XbaI digested plasmid tdTOM. Oligonucleotides containing a T2A peptide (primers 5 and 6) and an SV40 large T NLS triple sequence were hybridized and subcloned into a blunt pBluescript SK (+) plasmid. This plasmid was subsequently digested with NotI and KpnI and the T2A-NLS fragment was cloned into the tdTOM plasmid. The tdTOM was amplified by PCR from the initial plasmid (primers 7 and 8) adding KpnI and FseI sites and was subcloned into the donor vector after the 3xNLS sequence.

An FNF cassette containing two FRT sites and the NeoR / KanR gene under the control of the PGK promoter was amplified by PCR (primers 9 and 10) adding FseI and PacI sites. This fragment was subcloned into the donor vector. The highest scoring sgRNA sequence to target the STOP codon region of Myog was determined using a guideline design tool (crispr.mit.edu, Zhang Lab). The primers containing this targeting sequence (primers 11 and 12) were hybridized and subcloned into the BbsI-digested vector pU6- (BbsI) CBh-Cas9-T2A-mCherry.

3. Mouse embryonic stem cell selection

The donor constructs Myog-ntdTOM (linearized by digestion with PvuI) and vector pU6 were electroporated into C57BL / 6J mouse embryonic stem cells. After selection for G418 (300 μg / ml), positive clones were determined by PCR using primers 13, 14, and 15 (Supplementary Table 1), which produced a 1.7 kb band for WT and 1, 2 kb for the mutant. Two positive clones were expanded and 8 to 10 embryonic stem cells were injected into BALB / c blastocysts to generate chimeric mice at the Institut Pasteur’s mouse genetic engineering facility.

Germline transmission was verified by PCR and F1 mice were crossed with Tg (ACTFLPe) 9205Dym animals to excise the FNF cassette. The excision of the FNF cassette and the presence of the MyogntdTom allele was verified by PCR using primers 16, 17, and 18 (Supplementary Table 1) (MyogntdTom allele recombined with Flp, 236 bp, and WT 600 bp allele, Figure S1), and these primers they were later used for genotyping. F2 animals were backcrossed with C57BL / 6 animals to eliminate the Tg allele (ACTFLPe), and MyogntdTom / + animals were selected for further characterization.