STOCK Gt(ROSA)26Sor^{tm1(rtTA,EGFP)Nagy} TgTn(pb-tetO-Pou5f1,-Klf4,-Myc,-Sox2,-mCherry)250Nagy Tg(Pou5f1-EGFP)1Nagy/J

Stock number: 031010
Product name: Cre-inducible iRep1 [also called Oct4-GFP; ROSA26-rtTA(neo); OKMSCh250]
Research areas: Cancer Research, Cell Biology Research, Research Tools

Description

Cre-inducible iRep1 mice, also called Oct4-GFP; ROSA26-rtTA(neo); OKMSCh250, have the Oct4-GFP transgene, the Cre-inducible ROSA26-rtTA(neo) allele and the TetO-OKMS-mCherry transgene from line 250 (OKMSCh250). Following Cre recombinase exposure, these 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.
Of note, Cre-inducible iRep1 mice without the Oct4-GFP transgene are available as Stock No. 031012.

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; described below), 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. 031010: Cre-inducible iRep1 mice, also called Oct4-GFP; ROSA26-rtTA(neo); OKMSCh250, harbor the Oct4-GFP transgene, the Cre-inducible ROSA26-rtTA(neo) allele and the TetO-OKMS-mCherry transgene from line 250 (OKMSCh250).
The ROSA26-rtTA(neo) allele has a floxed PGK-neo-pA cassette preventing transcription of the downstream reverse tetracycline controlled transactivator (rtTA) sequences. 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.
Exposure to Cre recombinase results in functional rtTA activity from the ROSA26-rtTA(neo) allele (see below for GFP expression). Further administration of the tetracycline analog doxycycline (dox) results in mCherry fluorescence and expression of the OKMS transcription factors from the TetO-OKMS-mCherry transgene - which reprograms somatic cells that are derived from these animals to embryonic stem cell-like induced pluripotent stem cells (iPSCs).
The donating investigator reports that cells derived from iRep1 animals exhibit no OKMS or mCherry expression prior to dox administration.
The Oct4-GFP transgene has EGFP expression directed by mouse Oct4 promoter sequences, and is Tet-independent. As EGFP fluorescence coincides with expression of endogenous OCT4, this allows fluorescent determination of iPSC reprogramming/pluripotency status.
Prior to Cre administration, the donating investigator reports no rtTA or GFP expression is observed from the ROSA26-rtTA(neo) allele. Exposure to Cre recombinase results in functional rtTA activity and weak expression of the downstream IRES-EGFP (not visible using standard fluorescent microscopy, and can be distinguished from Oct4-GFP using flow cytometry).
Prior to Cre and dox administration, when Cre-inducible iRep1 mice are homozygous for ROSA26-rtTA(neo), homozygous for OKMSCh250 and carrying at least one copy of the Oct4-GFP transgene (rtTA(neo)^tm/tm; OKMS^tg/tg GFP^tg/0), they are viable and fertile with no reported abnormalities. Additionally, the donating investigator observes no significant phenotype differences between animals homozygous or hemizygous for the Oct4-GFP 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

Cre-inducible iRep1 mice, also called Oct4-GFP; ROSA26-rtTA(neo); OKMSCh250, have the Oct4-GFP transgene, the Cre-inducible ROSA26-rtTA(neo) allele and the TetO-OKMS-mCherry transgene from line 250 (OKMSCh250). Each mutation/transgene was designed in the Dr. Andras Nagy laboratory (Lunenfeld-Tanenbaum Research Institute [LTRI], Mount Sinai Hospital), and is described below.

The Cre-inducible ROSA26-rtTA(neo) allele (Gt(ROSA)26Sor^{tm1(rtTA,EGFP)Nagy}) has a loxP::PGK-neo-pA::loxP cassette, the reverse tetracycline-controlled transactivator gene (rtTA), an internal ribosomal entry site (IRES) and an enhanced green fluorescent protein gene (EGFP) with polyA sequence inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus on chromosome 6. Mice derived from C57BL/6J-congenic ROSA26-rtTA(neo) (Stock No. 005670) were used as described below.

The Oct4-GFP transgene (Tg(Pou5f1-EGFP)1Nagy) has an 18 kbp mouse Pou5f1 fragment (Oct4; including ~1.8 kbp of upstream sequences) with an EGFP gene inserted immediately adjacent to the Pou5f1 initiator codon. This was co-electroporated with a transient PGK-puro plasmid (see below) into 129X1/SvJ x 129S1/Sv)F1-Kitl⁺-derived R1 embryonic stem (ES) cells, and ES cells with desired regulation of GFP expression were selected. Founders were bred to Crl:CD-1 (ICR) mice for germline transmission. Oct4-GFP transgenic mice (ICR;129 genetic background [CD-1 x 129S2]) were the source of mouse embryonic fibroblasts (MEFs) used below. The Oct4-GFP transgene insertion site(s) and copy number are unknown (December 2017). The Oct4-GFP transgene is segregating with puro (September 2018).

ICR;129 Oct4-GFP MEFs 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 #F7 (interrupted for 48 hours) were bred to C57BL/6J mice. Germline transmitting mice were bred with C57BL/6J to establish founder line OKMSCh250 (harboring a single transgene insertion on chromosome 14:97,485,545-97,485,549[mm9]).
Next, Oct4-GFP; OKMSCh250 mice were bred to C57BL/6J-congenic ROSA26-rtTA(neo) mice. The donating investigator reports that the resulting Oct4-GFP; ROSA26-rtTA(neo); OKMSCh250 colony (Cre-inducible iRep1) 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 coat color were sent to The Jackson Laboratory. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

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 11, 12 and 18 were segregating for C57BL/6 and 129 (or other) allele-types, 1 marker on chromosome 19 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 ~88-92% 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.

Allele

Symbol: Gt(ROSA)26Sor^{tm1(rtTA,EGFP)Nagy}
Name: targeted mutation 1, Andras Nagy
MGI accession ID: MGI:3583817
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Transactivator
Synonyms: Gt(ROSA)26Sor^{tm1(rtTA)Nagy}; Gt(ROSA)26Sor^tm1Nagy; R26^rtTA; Rosa26^fs-rtTA; ROSA26-rtTA; ROSA26-rtTA-IRES-EGFP; Rosa-eGFP; Rosa-rtTA^LSL; rtTA^flox
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Expressed genes: rtTA,reverse tetracycline-controlled transactivator,E. coli; GFP,Green Fluorescent Protein,
Site of expression: When these mutant mice are crossed to a strain expressing Cre recombinase, rtTA and EGFP are expressed in the cre-expressing tissues. These mice can be used to generate mutant mice in which the tissue specificity of cre-expression and doxycycline inducibility of the rtTA can be combined.
Molecular note: A targeting vector containing a floxed Pgk-neo-pA cassette and a rtTA-IRES-EGFP-pA cassette was inserted into intron 1 of the locus. Upon cre expression, the floxed neo cassette is removed and the ROSA26 promoter drives expression of rtTA and EGFP. Presence of doxycycline results in the formation of an active transcriptional activator and the activation of the responder transgene.

Allele

Symbol: Tg(Pou5f1-EGFP)1Nagy
Name: transgene insertion 1, Andras Nagy
MGI accession ID: MGI:5902938
Generation method: Transgenic
Attributes: Reporter
Synonyms: Oct4-GFP
Marker symbol: Tg(Pou5f1-EGFP)1Nagy
Marker name: transgene insertion 1, Andras Nagy
Chromosome: UN
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Molecular note: This transgene allows EGFP expression driven by the Oct4 promoter.

Allele

Symbol: TgTn(pb-tetO-Pou5f1,-Klf4,-Myc,-Sox2,-mCherry)250Nagy
Name: transgenic transposon concatemer 250, Andras Nagy
MGI accession ID: MGI:6507139
Generation method: Transgenic
Attributes: Reporter; Transposon concatemer; Inserted expressed sequence
Synonyms: OKMSCh250
Marker symbol: TgTn(pb-tetO-Pou5f1,-Klf4,-Myc,-Sox2,-mCherry)250Nagy
Marker name: transgenic transposan concatemer 250, Andras Nagy
Chromosome: 14
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 250 harbors a single transgene insertion on chromosome 14:97,485,545-97,485,549[mm9]).