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

Stock number: 031012
Product name: Cre-inducible iRep1 without Oct4-GFP [also called ROSA26-rtTA(neo); OKMSCh250]
Research areas: Cancer Research, Cell Biology Research, Research Tools

Description

Cre-inducible iRep1 without Oct4-GFP mice, also called ROSA26-rtTA(neo); OKMSCh250, have 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.
Of note, Cre-inducible iRep1 mice with the Oct4-GFP transgene are available as Stock No. 031010.

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; described below) and the OKMSCh72 transgenic mice (Stock No. 031009) useful for creating iRep2.

Stock No. 031012: Cre-inducible iRep1 without Oct4-GFP mice, also called ROSA26-rtTA(neo); OKMSCh250, harbor 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.
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).
Prior to Cre and dox administration, when Cre-inducible iRep1 without Oct4-GFP mice are homozygous for ROSA26-rtTA(neo) and homozygous for OKMSCh250 (rtTA(neo)^tm/tm; OKMS^tg/tg), they are viable and fertile with no reported abnormalities.

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 without Oct4-GFP mice, also called ROSA26-rtTA(neo); OKMSCh250, have 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).

In 2018, Dr. Nagy sent Cre-inducible iRep1 mice with the Oct4-GFP transgene (Tg(Pou5f1-EGFP)1Nagy) on ~88-92% C57BL/6 genetic background to The Jackson Laboratory as Stock No. 031010. Upon arrival, those were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation. Resulting mice harboring only the Cre-inducible ROSA26-rtTA(neo) allele and the OKMSCh250 transgene were used to establish our live colony of Cre-inducible iRep1 without Oct4-GFP mice as Stock No. 031012.

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: 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]).