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Discovery of a novel murine keratin 6 (K6) isoform explains the absence of hair and nail defects in mice deficient for K6a and K6b.

Wojcik SM, Longley MA, Roop DR - J. Cell Biol. (2001)

Bottom Line: We cloned this previously unknown murine keratin gene and found it to be highly homologous to human K6hf, which is expressed in hair follicles.We therefore termed this gene MK6 hair follicle (MK6hf).The presence of MK6hf in the MK6a/b-/- follicles and nails offers an explanation for the absence of hair and nail defects in MK6a/b-/- animals.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
The murine genome is known to have two keratin 6 (K6) genes, mouse K6 (MK6)a and MK6b. These genes display a complex expression pattern with constitutive expression in the epithelia of oral mucosa, hair follicles, and nail beds. We generated mice deficient for both genes through embryonic stem cell technology. The majority of MK6a/b-/- mice die of starvation within the first two weeks of life. This is due to a localized disintegration of the dorsal tongue epithelium, which results in the build up of a plaque of cell debris that severely impairs feeding. However, approximately 25% of MK6a/b-/- mice survive to adulthood. Remarkably, the surviving MK6a/b-/- mice have normal hair and nails. To our surprise, we discovered MK6 staining both in the hair follicle and the nail bed of MK6a/b-/- mice, indicating the presence of a third MK6 gene. We cloned this previously unknown murine keratin gene and found it to be highly homologous to human K6hf, which is expressed in hair follicles. We therefore termed this gene MK6 hair follicle (MK6hf). The presence of MK6hf in the MK6a/b-/- follicles and nails offers an explanation for the absence of hair and nail defects in MK6a/b-/- animals.

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Targeting of MK6a and MK6b. (a) A replacement vector containing the hprtΔ3′-neo cassette was used to delete MK6a, MK6b, and the intergenic region. The positions of the 5′ and 3′ external probes are indicated by short bars. The triangles indicate the position of the PCR primers used for genotyping. (b) Genomic Southerns: 5′ Southern, EcoRV (RV) digest, showing the wild-type 9-kb and the targeted 7-kb fragments detected with the 5′ external probe; 3′ Southern, SphI (S) digest, showing the 10-kb wild-type and 7.5-kb targeted allele. (c) Genomic PCR of the 3′ end of the targeted locus showing a 475-bp product for the MK6a/b+/+ genotype, a 245-bp product for MK6a/b−/−, and both fragments for MK6a/b+/−. (d) RNase protection assay of RNA isolated from uninduced skin with anagen hair follicles. Both MK6a and MK6b mRNAs are absent in the MK6a/b−/− sample. The probe was designed to protect 382 bases in MK6a and 268 bases in MK6b. Please note that two bands are generated for the MK6a transcript, most likely due to a polymorphism present in the C57BL6/N strain, resulting in partial degradation of the 382-bp fragment. A cyclophilin probe was used as a loading control.
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fig1: Targeting of MK6a and MK6b. (a) A replacement vector containing the hprtΔ3′-neo cassette was used to delete MK6a, MK6b, and the intergenic region. The positions of the 5′ and 3′ external probes are indicated by short bars. The triangles indicate the position of the PCR primers used for genotyping. (b) Genomic Southerns: 5′ Southern, EcoRV (RV) digest, showing the wild-type 9-kb and the targeted 7-kb fragments detected with the 5′ external probe; 3′ Southern, SphI (S) digest, showing the 10-kb wild-type and 7.5-kb targeted allele. (c) Genomic PCR of the 3′ end of the targeted locus showing a 475-bp product for the MK6a/b+/+ genotype, a 245-bp product for MK6a/b−/−, and both fragments for MK6a/b+/−. (d) RNase protection assay of RNA isolated from uninduced skin with anagen hair follicles. Both MK6a and MK6b mRNAs are absent in the MK6a/b−/− sample. The probe was designed to protect 382 bases in MK6a and 268 bases in MK6b. Please note that two bands are generated for the MK6a transcript, most likely due to a polymorphism present in the C57BL6/N strain, resulting in partial degradation of the 382-bp fragment. A cyclophilin probe was used as a loading control.

Mentions: The MK6a/b targeting vector was designed to delete the complete coding regions of the MK6a and MK6b genes as well as the intervening 10.5 kb, resulting in a deletion of ∼18 kb (Fig. 1 a). The targeting vector was introduced into 129/SvEv AB2.2 embryonic stem (ES) cells. Successfully targeted clones were confirmed by Southern analysis using 5′ and 3′ external probes (Fig. 1 b). Targeted clones were injected into C57BL/6N blastocysts, and the chimeras were bred to C57BL/6N females. Germline transmission was obtained from one clone, and the first F1 MK6a/b+/− males were crossed with wild-type C57BL/6N females. The resulting MK6a/b+/− F2 animals were intercrossed, and their offspring were used for all analyses performed in this study. The absence of MK6a and MK6b transcripts was confirmed by RNase protection analysis (Fig. 1 d).


Discovery of a novel murine keratin 6 (K6) isoform explains the absence of hair and nail defects in mice deficient for K6a and K6b.

Wojcik SM, Longley MA, Roop DR - J. Cell Biol. (2001)

Targeting of MK6a and MK6b. (a) A replacement vector containing the hprtΔ3′-neo cassette was used to delete MK6a, MK6b, and the intergenic region. The positions of the 5′ and 3′ external probes are indicated by short bars. The triangles indicate the position of the PCR primers used for genotyping. (b) Genomic Southerns: 5′ Southern, EcoRV (RV) digest, showing the wild-type 9-kb and the targeted 7-kb fragments detected with the 5′ external probe; 3′ Southern, SphI (S) digest, showing the 10-kb wild-type and 7.5-kb targeted allele. (c) Genomic PCR of the 3′ end of the targeted locus showing a 475-bp product for the MK6a/b+/+ genotype, a 245-bp product for MK6a/b−/−, and both fragments for MK6a/b+/−. (d) RNase protection assay of RNA isolated from uninduced skin with anagen hair follicles. Both MK6a and MK6b mRNAs are absent in the MK6a/b−/− sample. The probe was designed to protect 382 bases in MK6a and 268 bases in MK6b. Please note that two bands are generated for the MK6a transcript, most likely due to a polymorphism present in the C57BL6/N strain, resulting in partial degradation of the 382-bp fragment. A cyclophilin probe was used as a loading control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196416&req=5

fig1: Targeting of MK6a and MK6b. (a) A replacement vector containing the hprtΔ3′-neo cassette was used to delete MK6a, MK6b, and the intergenic region. The positions of the 5′ and 3′ external probes are indicated by short bars. The triangles indicate the position of the PCR primers used for genotyping. (b) Genomic Southerns: 5′ Southern, EcoRV (RV) digest, showing the wild-type 9-kb and the targeted 7-kb fragments detected with the 5′ external probe; 3′ Southern, SphI (S) digest, showing the 10-kb wild-type and 7.5-kb targeted allele. (c) Genomic PCR of the 3′ end of the targeted locus showing a 475-bp product for the MK6a/b+/+ genotype, a 245-bp product for MK6a/b−/−, and both fragments for MK6a/b+/−. (d) RNase protection assay of RNA isolated from uninduced skin with anagen hair follicles. Both MK6a and MK6b mRNAs are absent in the MK6a/b−/− sample. The probe was designed to protect 382 bases in MK6a and 268 bases in MK6b. Please note that two bands are generated for the MK6a transcript, most likely due to a polymorphism present in the C57BL6/N strain, resulting in partial degradation of the 382-bp fragment. A cyclophilin probe was used as a loading control.
Mentions: The MK6a/b targeting vector was designed to delete the complete coding regions of the MK6a and MK6b genes as well as the intervening 10.5 kb, resulting in a deletion of ∼18 kb (Fig. 1 a). The targeting vector was introduced into 129/SvEv AB2.2 embryonic stem (ES) cells. Successfully targeted clones were confirmed by Southern analysis using 5′ and 3′ external probes (Fig. 1 b). Targeted clones were injected into C57BL/6N blastocysts, and the chimeras were bred to C57BL/6N females. Germline transmission was obtained from one clone, and the first F1 MK6a/b+/− males were crossed with wild-type C57BL/6N females. The resulting MK6a/b+/− F2 animals were intercrossed, and their offspring were used for all analyses performed in this study. The absence of MK6a and MK6b transcripts was confirmed by RNase protection analysis (Fig. 1 d).

Bottom Line: We cloned this previously unknown murine keratin gene and found it to be highly homologous to human K6hf, which is expressed in hair follicles.We therefore termed this gene MK6 hair follicle (MK6hf).The presence of MK6hf in the MK6a/b-/- follicles and nails offers an explanation for the absence of hair and nail defects in MK6a/b-/- animals.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
The murine genome is known to have two keratin 6 (K6) genes, mouse K6 (MK6)a and MK6b. These genes display a complex expression pattern with constitutive expression in the epithelia of oral mucosa, hair follicles, and nail beds. We generated mice deficient for both genes through embryonic stem cell technology. The majority of MK6a/b-/- mice die of starvation within the first two weeks of life. This is due to a localized disintegration of the dorsal tongue epithelium, which results in the build up of a plaque of cell debris that severely impairs feeding. However, approximately 25% of MK6a/b-/- mice survive to adulthood. Remarkably, the surviving MK6a/b-/- mice have normal hair and nails. To our surprise, we discovered MK6 staining both in the hair follicle and the nail bed of MK6a/b-/- mice, indicating the presence of a third MK6 gene. We cloned this previously unknown murine keratin gene and found it to be highly homologous to human K6hf, which is expressed in hair follicles. We therefore termed this gene MK6 hair follicle (MK6hf). The presence of MK6hf in the MK6a/b-/- follicles and nails offers an explanation for the absence of hair and nail defects in MK6a/b-/- animals.

Show MeSH
Related in: MedlinePlus