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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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“Reprogrammable mice” carrying single copies of reprogramming factorsA) Experimental outline. iB-iPS#9 chimera10 is mated to generate offspring with different transgene copy number. Blood and tail fibroblasts were collected from adult offspring and MEF cultures were established from day E13.5 embryos. B) Southern analysis of iBiPS#9 line and V6.5 ESCs as controls. Filled arrowheads: endogenous bands; open arrowheads: proviral integrations. C) Top Panels: iPS colony formation from F1 offspring 9.27 (O1S1K1M1). Immuno-fluorescent analysis of the same iPS cell line that grew independently of Dox is shown in the lower panel. D) Southern analysis of F1 progeny blood derived iPS lines *: non-specific background bands. E) iPS cells contribute to chimeras (black arrow) that exhibit germline transmission (transgenic offspring: white arrows). F) Reprogramming efficiency of CD11b+ cells, 28 days after Dox induction. Efficiencies calculated as the fraction of Nanog positive colonies to cells seeded. Error bars: SD in duplicate wells. The generation (F1 or F2) and transgene copy number (subscript) are shown. “B” indicates iPS-line derived from peripheral blood.
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Figure 1: “Reprogrammable mice” carrying single copies of reprogramming factorsA) Experimental outline. iB-iPS#9 chimera10 is mated to generate offspring with different transgene copy number. Blood and tail fibroblasts were collected from adult offspring and MEF cultures were established from day E13.5 embryos. B) Southern analysis of iBiPS#9 line and V6.5 ESCs as controls. Filled arrowheads: endogenous bands; open arrowheads: proviral integrations. C) Top Panels: iPS colony formation from F1 offspring 9.27 (O1S1K1M1). Immuno-fluorescent analysis of the same iPS cell line that grew independently of Dox is shown in the lower panel. D) Southern analysis of F1 progeny blood derived iPS lines *: non-specific background bands. E) iPS cells contribute to chimeras (black arrow) that exhibit germline transmission (transgenic offspring: white arrows). F) Reprogramming efficiency of CD11b+ cells, 28 days after Dox induction. Efficiencies calculated as the fraction of Nanog positive colonies to cells seeded. Error bars: SD in duplicate wells. The generation (F1 or F2) and transgene copy number (subscript) are shown. “B” indicates iPS-line derived from peripheral blood.

Mentions: Screening approaches using infected cells are hampered by the genetic variability caused by the random integrations of multiple proviral copies9,10. Recently, we generated a “secondary” transgenic system that eliminates such heterogeneity9,10. In this approach, mouse embryonic fibroblasts (MEFs) heterozygous for the ROSA26-M2 reverse tetracycline transactivator (M2-rtTA) were infected with Doxycycline (Dox)-inducible lentiviruses carrying the four reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) and induced to generate “primary” iPS cells by addition of Dox. These cells were used to obtain chimeric mice with genetically identical somatic cells that can be isolated and reprogrammed in vitro by addition of Dox. However, such “secondary” somatic cells require isolation from chimeric mice and contain copies of all four factors required for reprogramming, thus impeding their use in drug screens aimed at identifying components that can substitute for a given transcription factor. Here we describe the generation of genetically homogeneous mice and MEF lines containing different combinations of a defined set of Dox-inducible proviral genomes. This was achieved through random segregation of the integrated lentiviruses after germline transmission from “primary” iPS-derived chimeras (Fig.1A). We used the previously described Pro B cell-derived iB-iPS#9 cell line10 which carried a single c-Myc and Sox2 and two Klf4 and Oct4 proviral copies, respectively (O2S1K2M1) (Fig.1B, Supplementary Fig.1 online). To produce transgenic offspring an iB-iPS#9 chimera that transmitted the transgenes through the germline in 100% of the offspring was crossed to wild-type females (Fig.1A) and 91 individual offspring were genotyped. This analysis indentified mice carrying all possible combinations of one, two, three or all four vectors (supplementary Fig.2 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)

“Reprogrammable mice” carrying single copies of reprogramming factorsA) Experimental outline. iB-iPS#9 chimera10 is mated to generate offspring with different transgene copy number. Blood and tail fibroblasts were collected from adult offspring and MEF cultures were established from day E13.5 embryos. B) Southern analysis of iBiPS#9 line and V6.5 ESCs as controls. Filled arrowheads: endogenous bands; open arrowheads: proviral integrations. C) Top Panels: iPS colony formation from F1 offspring 9.27 (O1S1K1M1). Immuno-fluorescent analysis of the same iPS cell line that grew independently of Dox is shown in the lower panel. D) Southern analysis of F1 progeny blood derived iPS lines *: non-specific background bands. E) iPS cells contribute to chimeras (black arrow) that exhibit germline transmission (transgenic offspring: white arrows). F) Reprogramming efficiency of CD11b+ cells, 28 days after Dox induction. Efficiencies calculated as the fraction of Nanog positive colonies to cells seeded. Error bars: SD in duplicate wells. The generation (F1 or F2) and transgene copy number (subscript) are shown. “B” indicates iPS-line derived from peripheral blood.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654270&req=5

Figure 1: “Reprogrammable mice” carrying single copies of reprogramming factorsA) Experimental outline. iB-iPS#9 chimera10 is mated to generate offspring with different transgene copy number. Blood and tail fibroblasts were collected from adult offspring and MEF cultures were established from day E13.5 embryos. B) Southern analysis of iBiPS#9 line and V6.5 ESCs as controls. Filled arrowheads: endogenous bands; open arrowheads: proviral integrations. C) Top Panels: iPS colony formation from F1 offspring 9.27 (O1S1K1M1). Immuno-fluorescent analysis of the same iPS cell line that grew independently of Dox is shown in the lower panel. D) Southern analysis of F1 progeny blood derived iPS lines *: non-specific background bands. E) iPS cells contribute to chimeras (black arrow) that exhibit germline transmission (transgenic offspring: white arrows). F) Reprogramming efficiency of CD11b+ cells, 28 days after Dox induction. Efficiencies calculated as the fraction of Nanog positive colonies to cells seeded. Error bars: SD in duplicate wells. The generation (F1 or F2) and transgene copy number (subscript) are shown. “B” indicates iPS-line derived from peripheral blood.
Mentions: Screening approaches using infected cells are hampered by the genetic variability caused by the random integrations of multiple proviral copies9,10. Recently, we generated a “secondary” transgenic system that eliminates such heterogeneity9,10. In this approach, mouse embryonic fibroblasts (MEFs) heterozygous for the ROSA26-M2 reverse tetracycline transactivator (M2-rtTA) were infected with Doxycycline (Dox)-inducible lentiviruses carrying the four reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) and induced to generate “primary” iPS cells by addition of Dox. These cells were used to obtain chimeric mice with genetically identical somatic cells that can be isolated and reprogrammed in vitro by addition of Dox. However, such “secondary” somatic cells require isolation from chimeric mice and contain copies of all four factors required for reprogramming, thus impeding their use in drug screens aimed at identifying components that can substitute for a given transcription factor. Here we describe the generation of genetically homogeneous mice and MEF lines containing different combinations of a defined set of Dox-inducible proviral genomes. This was achieved through random segregation of the integrated lentiviruses after germline transmission from “primary” iPS-derived chimeras (Fig.1A). We used the previously described Pro B cell-derived iB-iPS#9 cell line10 which carried a single c-Myc and Sox2 and two Klf4 and Oct4 proviral copies, respectively (O2S1K2M1) (Fig.1B, Supplementary Fig.1 online). To produce transgenic offspring an iB-iPS#9 chimera that transmitted the transgenes through the germline in 100% of the offspring was crossed to wild-type females (Fig.1A) and 91 individual offspring were genotyped. This analysis indentified mice carrying all possible combinations of one, two, three or all four vectors (supplementary Fig.2 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