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Transgenic mice with defined combinations of drug-inducible reprogramming factors.

Markoulaki S, Hanna J, Beard C, Carey BW, Cheng AW, Lengner CJ, Dausman JA, Fu D, Gao Q, Wu S, Cassady JP, Jaenisch R - Nat. Biotechnol. (2009)

Bottom Line: Proviruses carrying drug-inducible Oct4, Sox2, Klf4 and c-Myc used to derive 'primary' induced pluripotent stem (iPS) cells were segregated through germline transmission, generating mice and cells carrying subsets of the reprogramming factors.Drug treatment produced 'secondary' iPS cells only when the missing factor was introduced.This approach creates a defined system for studying reprogramming mechanisms and allows screening of genetically homogeneous cells for compounds that can replace any transcription factor required for iPS cell derivation.

View Article: PubMed Central - PubMed

Affiliation: The Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA.

ABSTRACT
Proviruses carrying drug-inducible Oct4, Sox2, Klf4 and c-Myc used to derive 'primary' induced pluripotent stem (iPS) cells were segregated through germline transmission, generating mice and cells carrying subsets of the reprogramming factors. Drug treatment produced 'secondary' iPS cells only when the missing factor was introduced. This approach creates a defined system for studying reprogramming mechanisms and allows screening of genetically homogeneous cells for compounds that can replace any transcription factor required for iPS cell derivation.

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MEF line library carrying different combinations of reprogramming factorsA) PCR genotyping of select independent M2-rtTa+ MEF lines from mating offspring 9.27 (O1S1K1M1) to wild-type females. Genotype is indicated at the bottom. B) iPS cell derivation from MEF lines carrying three or more factor combinations. Missing factor was introduced by infection with TetO-FUW lentivirus (FUW) carrying the missing transcription factor. NA: not applicable, ND: not determined. The efficiencies reported are based on Nanog+ colonies fixed 30 days after plating 10,000 cells and addition of Dox. C) iPS cells from three factor MEF lines lacking c-Myc after transduction with Klf4. 200,000 O1S1K1 MEFs were infected with the indicated control virus and cultured in the present of Dox without passaging. Image of primary colony on Day 42 of Dox induction after infection with FUW-Klf4. Primary colonies were picked and passaged without Dox and expressed Nanog. Nine independent lines derived from two experiments. D) Kinetics of Nanog-GFP knock-in allele expression in two-factor lines, pre-treated or not with Dox, after transduction of the missing factors. 20,000 infected cells were seeded per well. Two wells were harvested every 48 hours for detection of Nanog-GFP by FACS. Nanog-GFP was defined by achieving >0.8% GFP positive cells. Blue dashed line: day of infection (d0). Pretreatment with Dox was done for 16 days. Two independent experimental sets are shown. Efficiency was determined after 28 days of Dox treatment as number of Nanog-GFP+ colonies per 10,000 cells initially seeded.
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Figure 2: MEF line library carrying different combinations of reprogramming factorsA) PCR genotyping of select independent M2-rtTa+ MEF lines from mating offspring 9.27 (O1S1K1M1) to wild-type females. Genotype is indicated at the bottom. B) iPS cell derivation from MEF lines carrying three or more factor combinations. Missing factor was introduced by infection with TetO-FUW lentivirus (FUW) carrying the missing transcription factor. NA: not applicable, ND: not determined. The efficiencies reported are based on Nanog+ colonies fixed 30 days after plating 10,000 cells and addition of Dox. C) iPS cells from three factor MEF lines lacking c-Myc after transduction with Klf4. 200,000 O1S1K1 MEFs were infected with the indicated control virus and cultured in the present of Dox without passaging. Image of primary colony on Day 42 of Dox induction after infection with FUW-Klf4. Primary colonies were picked and passaged without Dox and expressed Nanog. Nine independent lines derived from two experiments. D) Kinetics of Nanog-GFP knock-in allele expression in two-factor lines, pre-treated or not with Dox, after transduction of the missing factors. 20,000 infected cells were seeded per well. Two wells were harvested every 48 hours for detection of Nanog-GFP by FACS. Nanog-GFP was defined by achieving >0.8% GFP positive cells. Blue dashed line: day of infection (d0). Pretreatment with Dox was done for 16 days. Two independent experimental sets are shown. Efficiency was determined after 28 days of Dox treatment as number of Nanog-GFP+ colonies per 10,000 cells initially seeded.

Mentions: We generated somatic cell lines with different combinations of factors by crossing transgenic male 9.27 (O1S1K1M1; Fig.1D) with wild-type females. MEF cultures were established from individual embryos and genotyped for the segregated transgenes (Fig.2A). “Single-copy four-factor” (O1S1K1M1) MEF lines reproducibly generated iPS cells with approximately 1% efficiency (Fig.2B, Supplementary Fig.6 online). In contrast, no iPS colony formation was observed with “three-factor” lines i.e., OSK (n=3), OSM (n=2), SKM (n=1), OKM (n=3) (Fig.2B). However, when these MEF lines were transduced with the missing factor and grown in the presence of Dox, iPS colonies appeared within 14–21 days (Fig.2B and Supplementary Fig.S7 online) at efficiencies comparable to the highest reported for fibroblasts9. All lines grew Dox-independently, expressed pluripotency markers and induced teratomas in vivo (Fig.2B, Supplementary Fig.S8 online).


Transgenic mice with defined combinations of drug-inducible reprogramming factors.

Markoulaki S, Hanna J, Beard C, Carey BW, Cheng AW, Lengner CJ, Dausman JA, Fu D, Gao Q, Wu S, Cassady JP, Jaenisch R - Nat. Biotechnol. (2009)

MEF line library carrying different combinations of reprogramming factorsA) PCR genotyping of select independent M2-rtTa+ MEF lines from mating offspring 9.27 (O1S1K1M1) to wild-type females. Genotype is indicated at the bottom. B) iPS cell derivation from MEF lines carrying three or more factor combinations. Missing factor was introduced by infection with TetO-FUW lentivirus (FUW) carrying the missing transcription factor. NA: not applicable, ND: not determined. The efficiencies reported are based on Nanog+ colonies fixed 30 days after plating 10,000 cells and addition of Dox. C) iPS cells from three factor MEF lines lacking c-Myc after transduction with Klf4. 200,000 O1S1K1 MEFs were infected with the indicated control virus and cultured in the present of Dox without passaging. Image of primary colony on Day 42 of Dox induction after infection with FUW-Klf4. Primary colonies were picked and passaged without Dox and expressed Nanog. Nine independent lines derived from two experiments. D) Kinetics of Nanog-GFP knock-in allele expression in two-factor lines, pre-treated or not with Dox, after transduction of the missing factors. 20,000 infected cells were seeded per well. Two wells were harvested every 48 hours for detection of Nanog-GFP by FACS. Nanog-GFP was defined by achieving >0.8% GFP positive cells. Blue dashed line: day of infection (d0). Pretreatment with Dox was done for 16 days. Two independent experimental sets are shown. Efficiency was determined after 28 days of Dox treatment as number of Nanog-GFP+ colonies per 10,000 cells initially seeded.
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Related In: Results  -  Collection

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Figure 2: MEF line library carrying different combinations of reprogramming factorsA) PCR genotyping of select independent M2-rtTa+ MEF lines from mating offspring 9.27 (O1S1K1M1) to wild-type females. Genotype is indicated at the bottom. B) iPS cell derivation from MEF lines carrying three or more factor combinations. Missing factor was introduced by infection with TetO-FUW lentivirus (FUW) carrying the missing transcription factor. NA: not applicable, ND: not determined. The efficiencies reported are based on Nanog+ colonies fixed 30 days after plating 10,000 cells and addition of Dox. C) iPS cells from three factor MEF lines lacking c-Myc after transduction with Klf4. 200,000 O1S1K1 MEFs were infected with the indicated control virus and cultured in the present of Dox without passaging. Image of primary colony on Day 42 of Dox induction after infection with FUW-Klf4. Primary colonies were picked and passaged without Dox and expressed Nanog. Nine independent lines derived from two experiments. D) Kinetics of Nanog-GFP knock-in allele expression in two-factor lines, pre-treated or not with Dox, after transduction of the missing factors. 20,000 infected cells were seeded per well. Two wells were harvested every 48 hours for detection of Nanog-GFP by FACS. Nanog-GFP was defined by achieving >0.8% GFP positive cells. Blue dashed line: day of infection (d0). Pretreatment with Dox was done for 16 days. Two independent experimental sets are shown. Efficiency was determined after 28 days of Dox treatment as number of Nanog-GFP+ colonies per 10,000 cells initially seeded.
Mentions: We generated somatic cell lines with different combinations of factors by crossing transgenic male 9.27 (O1S1K1M1; Fig.1D) with wild-type females. MEF cultures were established from individual embryos and genotyped for the segregated transgenes (Fig.2A). “Single-copy four-factor” (O1S1K1M1) MEF lines reproducibly generated iPS cells with approximately 1% efficiency (Fig.2B, Supplementary Fig.6 online). In contrast, no iPS colony formation was observed with “three-factor” lines i.e., OSK (n=3), OSM (n=2), SKM (n=1), OKM (n=3) (Fig.2B). However, when these MEF lines were transduced with the missing factor and grown in the presence of Dox, iPS colonies appeared within 14–21 days (Fig.2B and Supplementary Fig.S7 online) at efficiencies comparable to the highest reported for fibroblasts9. All lines grew Dox-independently, expressed pluripotency markers and induced teratomas in vivo (Fig.2B, Supplementary Fig.S8 online).

Bottom Line: Proviruses carrying drug-inducible Oct4, Sox2, Klf4 and c-Myc used to derive 'primary' induced pluripotent stem (iPS) cells were segregated through germline transmission, generating mice and cells carrying subsets of the reprogramming factors.Drug treatment produced 'secondary' iPS cells only when the missing factor was introduced.This approach creates a defined system for studying reprogramming mechanisms and allows screening of genetically homogeneous cells for compounds that can replace any transcription factor required for iPS cell derivation.

View Article: PubMed Central - PubMed

Affiliation: The Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA.

ABSTRACT
Proviruses carrying drug-inducible Oct4, Sox2, Klf4 and c-Myc used to derive 'primary' induced pluripotent stem (iPS) cells were segregated through germline transmission, generating mice and cells carrying subsets of the reprogramming factors. Drug treatment produced 'secondary' iPS cells only when the missing factor was introduced. This approach creates a defined system for studying reprogramming mechanisms and allows screening of genetically homogeneous cells for compounds that can replace any transcription factor required for iPS cell derivation.

Show MeSH
Related in: MedlinePlus