T Miceribosomal subunit is indicated by a black bar. E. Coomassie blue SDS-PAGE gel and western blot of cytoskeletal extracts prepared from the bladder (lanes 1, 2, 3), lung (lanes 4, 5, 10781694 6) and colon (lanes 7, 8, 9) of wildtype (lanes 1, 4, 7), heterozygous (lanes 2, 4, 6) and homozygous (lanes 3, 6, 9) K7 get 115103-85-0 knockout mice probed with a C-terminal K7 antibody. M = molecular weight standards, sizes in kDa are as indicated. doi:10.1371/journal.pone.0064404.ghaematoxylin and eosin. Tissue sections were examined by a clinical pathologist experienced in the histological analysis of mouse tissues. For antibody staining, high-temperature antigen retrieval was performed overnight by incubating sections with 0.01 M citrate buffer. Endogenous peroxidase was quenched by 1 v/v H2O2 in PBS for 30 minutes. Sections were blocked with either goat or rabbit serum (10 v/v in PBS) depending on the host species of the secondary antibody. Primary antibodies were incubated for 1 hour at room temperature and were detected by HRP polymer anti-mouse or anti-rabbit Envision antibodies (DAKO). Sections were developed using DAB substrate (DAKO) then counterstained with Mayer’s haematoxylin.Ethical ConsiderationsAll work involving animals was approved by the University of Dundee ethical review committee and all scientific procedures were performed under project licence authority (to WHIM and EBL) from the Home Office in accordance with the Animals (Scientific Procedures) Act 1986.ResultsTo generate K7 knockout mice, we replaced exon 1 of the Krt7 gene and ,270 bp of the proximal promoter with a neomycin resistance cassette (Figure 1A) in order to prevent transcripts originating from this locus. Homozygous K7 knockout mice, on either the original 129P2/C57Bl6 mixed genetic background or those on the inbred C57Bl6 background, were born from heterozygous intercrosses at the expected Mendelian frequency of 1:2:1 indicating that the absence of K7 did not affect embryonic development. Homozygous K7 knockout mice were MedChemExpress 14636-12-5 phenotypically indistinguishable from heterozygous and wildtype littermates and both male and female homozygotes were fertile and reproduced normally. In the mouse, K7 is primarily expressed in various ductal and glandular epithelia and is highly expressed in transitional epithelia such as the bladder urothelium [2]. Semi-quantitative RT-PCR analysis of cDNA prepared from the bladder, lung, colon and kidney confirmed the absence of Krt7 transcripts in homozygous K7 knockout tissues (Figure 1C). Northern blotting of mRNA prepared from the bladder using the mouse K7 cDNA (exons 1?) as a probe showed the absence of any K7 mRNA transcripts in homozygous mice whereas in heterozygous mice there was approximately half the amount of Krt7 mRNA as compared to wildtype mice (Figure 1D). Western blotting of cytoskeletalenriched extracts prepared from the bladder, lung and colon of homozygous K7 knockout mice showed no K7 protein (Figure 1E). In heterozygous mice, there was an appreciable reduction in the amount of K7 protein as compared to cytoskeletal extracts prepared from wildtype tissues. Immunofluorescence microscopy of tissue cryosections using a polyclonal antibody raised against the C-terminus of murine K7 confirmed the absence of K7 protein in homozygous K7 knockout mice (Figure 2 and results not shown). In heterozygotes, K7 staining was comparable to that observed in wildtype tissues (results not shown). Overall, these series of experiments demonstrated that ou.T Miceribosomal subunit is indicated by a black bar. E. Coomassie blue SDS-PAGE gel and western blot of cytoskeletal extracts prepared from the bladder (lanes 1, 2, 3), lung (lanes 4, 5, 10781694 6) and colon (lanes 7, 8, 9) of wildtype (lanes 1, 4, 7), heterozygous (lanes 2, 4, 6) and homozygous (lanes 3, 6, 9) K7 knockout mice probed with a C-terminal K7 antibody. M = molecular weight standards, sizes in kDa are as indicated. doi:10.1371/journal.pone.0064404.ghaematoxylin and eosin. Tissue sections were examined by a clinical pathologist experienced in the histological analysis of mouse tissues. For antibody staining, high-temperature antigen retrieval was performed overnight by incubating sections with 0.01 M citrate buffer. Endogenous peroxidase was quenched by 1 v/v H2O2 in PBS for 30 minutes. Sections were blocked with either goat or rabbit serum (10 v/v in PBS) depending on the host species of the secondary antibody. Primary antibodies were incubated for 1 hour at room temperature and were detected by HRP polymer anti-mouse or anti-rabbit Envision antibodies (DAKO). Sections were developed using DAB substrate (DAKO) then counterstained with Mayer’s haematoxylin.Ethical ConsiderationsAll work involving animals was approved by the University of Dundee ethical review committee and all scientific procedures were performed under project licence authority (to WHIM and EBL) from the Home Office in accordance with the Animals (Scientific Procedures) Act 1986.ResultsTo generate K7 knockout mice, we replaced exon 1 of the Krt7 gene and ,270 bp of the proximal promoter with a neomycin resistance cassette (Figure 1A) in order to prevent transcripts originating from this locus. Homozygous K7 knockout mice, on either the original 129P2/C57Bl6 mixed genetic background or those on the inbred C57Bl6 background, were born from heterozygous intercrosses at the expected Mendelian frequency of 1:2:1 indicating that the absence of K7 did not affect embryonic development. Homozygous K7 knockout mice were phenotypically indistinguishable from heterozygous and wildtype littermates and both male and female homozygotes were fertile and reproduced normally. In the mouse, K7 is primarily expressed in various ductal and glandular epithelia and is highly expressed in transitional epithelia such as the bladder urothelium [2]. Semi-quantitative RT-PCR analysis of cDNA prepared from the bladder, lung, colon and kidney confirmed the absence of Krt7 transcripts in homozygous K7 knockout tissues (Figure 1C). Northern blotting of mRNA prepared from the bladder using the mouse K7 cDNA (exons 1?) as a probe showed the absence of any K7 mRNA transcripts in homozygous mice whereas in heterozygous mice there was approximately half the amount of Krt7 mRNA as compared to wildtype mice (Figure 1D). Western blotting of cytoskeletalenriched extracts prepared from the bladder, lung and colon of homozygous K7 knockout mice showed no K7 protein (Figure 1E). In heterozygous mice, there was an appreciable reduction in the amount of K7 protein as compared to cytoskeletal extracts prepared from wildtype tissues. Immunofluorescence microscopy of tissue cryosections using a polyclonal antibody raised against the C-terminus of murine K7 confirmed the absence of K7 protein in homozygous K7 knockout mice (Figure 2 and results not shown). In heterozygotes, K7 staining was comparable to that observed in wildtype tissues (results not shown). Overall, these series of experiments demonstrated that ou.
