STOCK TgTn(pb-tetO-Pou5f1,-Klf4,-Myc,-Sox2,-mCherry)72Nagy/J

Stock number: 031009
Product name: OKMSCh72
Research areas: Cell Biology Research, Research Tools

Description

OKMSCh72 mice have the TetO-OKMS-mCherry transgene from line 72. When bred to have the widespread rtTA-expressing ROSA26-rtTA(Δneo) allele and the Oct4-GFP transgene (see Stock No. 031011), the resulting iRep2 (inducible reprogrammable mouse 2) mice allow doxycycline (dox)-inducible expression of a polycistronic OKMS cassette encoding four transcription factors (Oct4 [Pou5f1], Klf4, c-Myc [Myc] and Sox2) - which reprograms somatic cells that are derived from these animals to embryonic stem cell-like induced pluripotent stem cells (iPSCs). In addition, OKMS expression is accompanied by mCherry fluorescence, and the Oct4-GFP transgene allows EGFP fluorescent determination of iPSC reprogramming/pluripotency status.

Detailed description

Dr. Andras Nagy has several doxycycline-inducible reprogrammable (iRep) mouse lines available from The Jackson Laboratory: iRep1 (Stock No. 031011), Cre-inducible iRep1 (Stock No. 031010), iRep1 without Oct4-GFP (Stock No. 031013), Cre-inducible iRep1 without Oct4-GFP (Stock No. 031012) and the OKMSCh72 transgenic mice (Stock No. 031009) useful for creating iRep2.

Stock No. 031009: OKMSCh72 mice have the TetO-OKMS-mCherry transgene from line 72. The TetO-OKMS-mCherry transgene contains a tetracycline responsive element-driven polycistronic OKMS cassette (encoding the Yamanaka transcription factors Oct4 [Pou5f1], Klf4, c-Myc [Myc] and Sox2) and an IRES-mCherry. In the presence of tetracycline-controlled transactivator protein (tTA) and/or reverse tetracycline-controlled transactivator protein (rtTA), expression of OKMS and mCherry can be regulated with tetracycline or its analog doxycycline (dox). OKMS expression reprograms somatic cells that are derived from these animals to embryonic stem cell-like induced pluripotent stem cells (iPSCs).

For example, breeding OKMSCh72 mice to have the widespread rtTA-expressing ROSA26-rtTA(Δneo) allele and the Oct4-GFP transgene (see Stock No. 031011) results in the triple mutant mouse line iRep2. Prior to dox administration, the donating investigator reported that cells derived from iRep2 animals exhibit no OKMS or mCherry. Whereas the donating investigator reported iRep2 mice homozygous for ROSA26-rtTA(Δneo), homozygous for OKMSCh72 and carrying at least one copy of the Oct4-GFP transgene (rtTA^tm/tm; OKMS^tg/tg GFP^tg/0) were viable and fertile with no reported abnormalities, The Jackson Laboratory live colony of triple mutant mice proved difficult to maintain as homozygous for ROSA26-rtTA(Δneo) and OKMSCh72, as well as homozygous for ROSA26-rtTA(Δneo) and hemizygous for OKMSCh72. As such, The Jackson Laboratory Stock No. 031009 colony is maintained as having only the OKMSCh72 transgene.

Both iRep1 and iRep2 allow a wide variety of adult and embryo tissues to have ESC-like colony forming ability and a dox-independence timeline (a fundamental parameter defining reprogramming efficiency) that is more efficient than previous systems. Specifically, both iRep1- and iRep2-derived cells show faster reprogramming and time to dox-independence than similarly-treated Col1a1-OSKM cells (see Stock No. 011004). For iRep1 and iRep2, reprogramming to dox-independence coincided with endogenous Oct4 activation. While no major differences in reprogramming speed or dox-independence times were observed between iRep1 and iRep2, some minor reprogramming efficiency differences were observed depending on tissue origin (see Elbaz et al. 2018 MSS ##:#### - Figure 2).

Development

Stock No. 031009 has the TetO-OKMS-mCherry transgene from line 72 (OKMSCh72) designed in the Dr. Andras Nagy laboratory (Lunenfeld-Tanenbaum Research Institute [LTRI], Mount Sinai Hospital).

First, MEFs from ICR;129 mice harboring the Oct4-GFP transgene (Tg(Pou5f1-EGFP)1Nagy; see Stock No. 031011) were transfected with three piggy-BAC transposon delivery constructs: PB:TetO-OKMS-mCherry (described below), PB:CAG-rtTA and pCMV-hyPBase (hyperactive transient transposase expression plasmid). PB:TetO-OKMS-mCherry contains a tetracycline responsive element (TRE; tetO), the polycistronic expression cassette OKMS, an IRES, the mCherry gene and polyA signal. The second generation TRE (SG-TRE) used here is tetO2 - containing a shortened CMV minimal promoter with eight copies of 19 bp tet operator sequences positioned in an optimized manner upstream of the TATA box. The OKMS cassette contains four mouse reprogramming genes - Oct4 (Pou5f1), Klf4, c-Myc (Myc) and Sox2 - each separated by viral 2A oligopeptides that mediate ribosomal skipping (F2A, E2A and F2A, respectively). Doxycycline-treated cells with overexpression of OKMS become reprogrammed to ES cell-like induced pluripotent stem cells (iPSCs), and those iPSCs were aggregated with ICR morulae. Chimeras generated from primary iPSC clone #72 (interrupted for 24 hours) were bred to C57BL/6J mice. Germline transmitting mice were bred with C57BL/6J to establish founder line OKMSCh72 (harboring a single transgene insertion into A530065N20Rik on chromosome 13:60,045,383-60,045,387[mm9] in transcriptionally reverse orientation).
Next, Oct4-GFP; OKMSCh72 mice were bred to C57BL/6J;C57BL/6N genetic background mice harboring the ROSA26-rtTA(Δneo) allele (Gt(ROSA)26Sor^{tm1.1(rtTA,EGFP)Nagy}; see Stock No. 031011). The donating investigator reports that the resulting Oct4-GFP; ROSA26-rtTA(Δneo); OKMSCh72 colony (iRep2) was backcrossed at least five generations to C57BL/6J (the randomly integrated PB:CAG-rtTA transgenes were removed) [see SNP results below]. In 2018, males with black or grey/light-agouti coat color were sent to The Jackson Laboratory. Upon arrival, mice were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation. Our live colony of triple mutant mice proved difficult to maintain as homozygous for ROSA26-rtTA(Δneo) and OKMSCh72, as well as homozygous for ROSA26-rtTA(Δneo) and hemizygous for OKMSCh72. As such, The Jackson Laboratory maintained Stock No. 031009 having only the OKMSCh72 transgene.

In 2018, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the males sent to The Jackson Laboratory Repository. This revealed several markers that were not fixed for C57BL/6 allele-type: single markers on chromosomes 2, 12, 15 and 16 were segregating for C57BL/6 and 129 (or other) allele-types, 1 marker on chromosome 8 was segregating for C57BL/6J and C57BL/6N (or other) allele-types, and 1 marker on chromosome 6 was segregating for C57BL/6J and 129 (or other) allele-types. Of note, the chromosome 6 marker is only ~6 Mbp away from Gt(ROSA)26Sor and may be indicative of the genetic background of the ES cells used to create that mutant allele). Collectively, these data suggest that the mice sent to The Jackson Laboratory Repository were ~86-90% C57BL/6 genetic background (which may be a mix of C57BL/6J;C57BL/6N), and may contain contributions from 129 and/or other genetic backgrounds. At least one additional generation of backcrossing to C57BL/6J was performed by The Jackson Laboratory Repository.

Allele

Symbol: TgTn(pb-tetO-Pou5f1,-Klf4,-Myc,-Sox2,-mCherry)72Nagy
Name: transgenic transposon concatemer 72, Andras Nagy
MGI accession ID: MGI:6506983
Generation method: Transgenic
Attributes: Reporter; Transposon concatemer; Inserted expressed sequence
Synonyms: OKMSCh250
Marker symbol: TgTn(pb-tetO-Pou5f1,-Klf4,-Myc,-Sox2,-mCherry)72Nagy
Marker name: transgenic transposan concatemer 72, Andras Nagy
Chromosome: 13
Strain of origin: 129 and ICR
Molecular note: The transgene consists of a piggy-BAC transposon delivery construct PB:TetO-OKMS-mCherry. PB:TetO-OKMS-mCherry contains a tetracycline responsive element (TRE; tetO), the polycistronic expression cassette OKMS, an IRES, the mCherry gene and polyA signal. The second generation TRE (SG-TRE) used here is tetO2 - containing a shortened CMV minimal promoter with eight copies of 19 bp tet operator sequences positioned in an optimized manner upstream of the TATA box. The OKMS cassette contains four mouse reprogramming genes - Oct4 (Pou5f1), Klf4, c-Myc (Myc) and Sox2 - each separated by viral 2A oligopeptides that mediate ribosomal skipping (F2A, E2A and F2A, respectively). Founder line 72 harbors a single transgene insertion into A530065N20Rik on chromosome 13:60,045,383-60,045,387[mm9] in transcriptionally reverse orientation.