PMC:1564426 / 18675-20927 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/1564426","sourcedb":"PMC","sourceid":"1564426","source_url":"http://www.ncbi.nlm.nih.gov/pmc/1564426","text":"Genotype- and Tissue-Specific Expression of the Truncated Apc Transcripts\nTo assess the molecular effects of the K14-cre–mediated recombination, we screened for the presence of deleted Apc (ApcΔ580) alleles. Genomic DNA was extracted from liver, thymus, and skin from all 4 possible genotypes: K14-cre; ApcCKO/CKO, K14-cre; ApcCKO/+, ApcCKO/CKO, and ApcCKO/+. Genotyping on genomic DNA from these tissues showed that the ApcΔ580 allele (500-bp product) was detected only from the skin and thymus of the K14-cre–positive mice. The presence of mutant Apc allele in the thymus of K14-cre; ApcCKO/+ mice was consistently much less than the DNA from the skin of the same animal or other tissues from the KA mutants. In addition, this product was not detected at all in either the liver of K14-cre–positive or in any of the K14-cre–negative mouse tissues samples, establishing that Cre-mediated recombination has taken place in the tissue-specific manner in the mice that inherited K14-cre (Figure 3A).\nFigure 3 Tissue-Specific Detection and Expression of Deleted Apc Alleles\n(A) Tissue-specific genotyping PCR. Only genomic DNA samples from the skin (S) and thymus (T), but not liver (L) of mice positive for K14-cre show the presence of deleted ApcΔ580 allele.\n(B) Genotype- and tissue-specific expression of the truncated Apc transcripts. A representative gel of RT-PCR using primers F546 and R721, showing that only RNA from the skin and thymus but not liver of mice positive for K14-cre have transcripts from both wild-type (528 bp) and deleted (313 bp) Apc alleles. Apc transcripts were also analyzed by RT-PCR with primers spanning exon 14 (Figure 1A) using total RNA isolated from the corresponding tissue samples. We detected the expected RT-PCR product (313 bp) from the truncated Apc (ApcΔ580) allele only in the tissues where Cre recombinase is known to be expressed in the K14-cre–positive mice. However, this product was not detected in either the K14-cre–negative mouse tissues samples or the liver of K14-cre–positive mice, and only the product from the wild-type allele (528 bp) was detected from these RNA samples, further confirming that Cre-mediated recombination has taken place in the tissue- and genotype-specific manner (Figure 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