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Genetic targeting of the endoderm with claudin-6CreER.

Anderson WJ, Zhou Q, Alcalde V, Kaneko OF, Blank LJ, Sherwood RI, Guseh JS, Rajagopal J, Melton DA - Dev. Dyn. (2008)

Bottom Line: The first step requires an understanding of definitive endoderm: the genes and signals responsible for its specification, proliferation, and patterning.To create a genetic system to drive gene expression throughout the definitive endoderm with both spatial and temporal control, we target the endogenous locus with an inducible Cre recombinase (Cre-ER(T2)) cassette.We also report a lineage analysis of the fate of Cldn6-expressing embryonic cells, which is relevant to the development of the pancreas, lung, and liver.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts 02138, USA.

ABSTRACT
A full description of the ontogeny of the beta cell would guide efforts to generate beta cells from embryonic stem cells (ESCs). The first step requires an understanding of definitive endoderm: the genes and signals responsible for its specification, proliferation, and patterning. This report describes a global marker of definitive endoderm, Claudin-6 (Cldn6). We report its expression in early development with particular attention to definitive endoderm derivatives. To create a genetic system to drive gene expression throughout the definitive endoderm with both spatial and temporal control, we target the endogenous locus with an inducible Cre recombinase (Cre-ER(T2)) cassette. Cldn6 mice are viable and fertile with no obvious phenotypic abnormalities. We also report a lineage analysis of the fate of Cldn6-expressing embryonic cells, which is relevant to the development of the pancreas, lung, and liver.

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Successful targeting of the Cldn6 locus with the CIHV cassette. A: Southern blot analysis. Upon successful recombination, the entire coding sequence for Cldn6 is removed, generating a  allele. After NcoI digest, the wild type locus generates a band of approximately 12.5 kb, while the targeted allele has a smaller band close to 8 kb. For reference, the first two bands on the 1-kb ladder are 10 and 8 kb, respectively. As seen in the parental ES cell lane (AV3), only one band is present higher than 10 kb, while in the targeted ES cell clone lane (E40), there is a band at the same height as in the wild type lane, but a second band present at 8 kb. B: Genotyping by PCR confirms germline transmission of the CIHV allele. AV3 and E40 refer to the parental ES cell line and targeted ES cell line used for blastocyst injection, respectively. The upper gel distinguishes between the wild type and targeted locus. The lower gel is a PCR to demonstrate ACN cassette removal during germline transmission. C,D: Cldn6CIHV/CIHV mice are  mutants for Cldn6. In situ hybridization for Cldn6 in embryos from a Cldn6CIHV/+ intercross. C: Cldn6CIHV/+; D: Cldn6CIHV/CIHV. As shown in D, Cldn6 mRNA is absent in the Cldn6CIHV/CIHV embryo.
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fig03: Successful targeting of the Cldn6 locus with the CIHV cassette. A: Southern blot analysis. Upon successful recombination, the entire coding sequence for Cldn6 is removed, generating a allele. After NcoI digest, the wild type locus generates a band of approximately 12.5 kb, while the targeted allele has a smaller band close to 8 kb. For reference, the first two bands on the 1-kb ladder are 10 and 8 kb, respectively. As seen in the parental ES cell lane (AV3), only one band is present higher than 10 kb, while in the targeted ES cell clone lane (E40), there is a band at the same height as in the wild type lane, but a second band present at 8 kb. B: Genotyping by PCR confirms germline transmission of the CIHV allele. AV3 and E40 refer to the parental ES cell line and targeted ES cell line used for blastocyst injection, respectively. The upper gel distinguishes between the wild type and targeted locus. The lower gel is a PCR to demonstrate ACN cassette removal during germline transmission. C,D: Cldn6CIHV/CIHV mice are mutants for Cldn6. In situ hybridization for Cldn6 in embryos from a Cldn6CIHV/+ intercross. C: Cldn6CIHV/+; D: Cldn6CIHV/CIHV. As shown in D, Cldn6 mRNA is absent in the Cldn6CIHV/CIHV embryo.

Mentions: AV3 mouse ES cells (a gift from Jill McMahon) were targeted with the CIHV cassette (Suppl. Fig. 3). Out of a total of 1,474 colonies screened, one clone (E40) successfully underwent homologous recombination at the Cldn6 locus (Fig. 3A,B). Chimeras were generated by blastocyst injection and transmitted the allele through the germline (Fig. 3B). Heterozygotes from this cross were renamed Cldn6CIHV/+.Fig. 3


Genetic targeting of the endoderm with claudin-6CreER.

Anderson WJ, Zhou Q, Alcalde V, Kaneko OF, Blank LJ, Sherwood RI, Guseh JS, Rajagopal J, Melton DA - Dev. Dyn. (2008)

Successful targeting of the Cldn6 locus with the CIHV cassette. A: Southern blot analysis. Upon successful recombination, the entire coding sequence for Cldn6 is removed, generating a  allele. After NcoI digest, the wild type locus generates a band of approximately 12.5 kb, while the targeted allele has a smaller band close to 8 kb. For reference, the first two bands on the 1-kb ladder are 10 and 8 kb, respectively. As seen in the parental ES cell lane (AV3), only one band is present higher than 10 kb, while in the targeted ES cell clone lane (E40), there is a band at the same height as in the wild type lane, but a second band present at 8 kb. B: Genotyping by PCR confirms germline transmission of the CIHV allele. AV3 and E40 refer to the parental ES cell line and targeted ES cell line used for blastocyst injection, respectively. The upper gel distinguishes between the wild type and targeted locus. The lower gel is a PCR to demonstrate ACN cassette removal during germline transmission. C,D: Cldn6CIHV/CIHV mice are  mutants for Cldn6. In situ hybridization for Cldn6 in embryos from a Cldn6CIHV/+ intercross. C: Cldn6CIHV/+; D: Cldn6CIHV/CIHV. As shown in D, Cldn6 mRNA is absent in the Cldn6CIHV/CIHV embryo.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2665265&req=5

fig03: Successful targeting of the Cldn6 locus with the CIHV cassette. A: Southern blot analysis. Upon successful recombination, the entire coding sequence for Cldn6 is removed, generating a allele. After NcoI digest, the wild type locus generates a band of approximately 12.5 kb, while the targeted allele has a smaller band close to 8 kb. For reference, the first two bands on the 1-kb ladder are 10 and 8 kb, respectively. As seen in the parental ES cell lane (AV3), only one band is present higher than 10 kb, while in the targeted ES cell clone lane (E40), there is a band at the same height as in the wild type lane, but a second band present at 8 kb. B: Genotyping by PCR confirms germline transmission of the CIHV allele. AV3 and E40 refer to the parental ES cell line and targeted ES cell line used for blastocyst injection, respectively. The upper gel distinguishes between the wild type and targeted locus. The lower gel is a PCR to demonstrate ACN cassette removal during germline transmission. C,D: Cldn6CIHV/CIHV mice are mutants for Cldn6. In situ hybridization for Cldn6 in embryos from a Cldn6CIHV/+ intercross. C: Cldn6CIHV/+; D: Cldn6CIHV/CIHV. As shown in D, Cldn6 mRNA is absent in the Cldn6CIHV/CIHV embryo.
Mentions: AV3 mouse ES cells (a gift from Jill McMahon) were targeted with the CIHV cassette (Suppl. Fig. 3). Out of a total of 1,474 colonies screened, one clone (E40) successfully underwent homologous recombination at the Cldn6 locus (Fig. 3A,B). Chimeras were generated by blastocyst injection and transmitted the allele through the germline (Fig. 3B). Heterozygotes from this cross were renamed Cldn6CIHV/+.Fig. 3

Bottom Line: The first step requires an understanding of definitive endoderm: the genes and signals responsible for its specification, proliferation, and patterning.To create a genetic system to drive gene expression throughout the definitive endoderm with both spatial and temporal control, we target the endogenous locus with an inducible Cre recombinase (Cre-ER(T2)) cassette.We also report a lineage analysis of the fate of Cldn6-expressing embryonic cells, which is relevant to the development of the pancreas, lung, and liver.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts 02138, USA.

ABSTRACT
A full description of the ontogeny of the beta cell would guide efforts to generate beta cells from embryonic stem cells (ESCs). The first step requires an understanding of definitive endoderm: the genes and signals responsible for its specification, proliferation, and patterning. This report describes a global marker of definitive endoderm, Claudin-6 (Cldn6). We report its expression in early development with particular attention to definitive endoderm derivatives. To create a genetic system to drive gene expression throughout the definitive endoderm with both spatial and temporal control, we target the endogenous locus with an inducible Cre recombinase (Cre-ER(T2)) cassette. Cldn6 mice are viable and fertile with no obvious phenotypic abnormalities. We also report a lineage analysis of the fate of Cldn6-expressing embryonic cells, which is relevant to the development of the pancreas, lung, and liver.

Show MeSH
Related in: MedlinePlus