Construction of rat tropoelastin in transgenic mice

来源 :延边大学医学学报 | 被引量 : 0次 | 上传用户:yigeyige
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OBJECTIVE To address the functional significance of tropoelastin isoforms, it was constructed three rat tropoelastin minigenes that constitutively encode different tropoelastin isoforms,including those isoforms that lack the domains encoded by exon 33 and exons 13~15. METHODS The transgenes identified as tropoprom 1,tropoprom 2,and tropoprom 3 were created from rat tropoelastin genomic and cDNA clones. Oligonucleotide primers specific for the rat tropoelastin transgene(TRANS7/8 5′ AGTCTCAACAGGTGCTGT 3′ and TRANS 16/17 5′ AGTTCCAGCGCCTGTAAT 3′) were utilized to amplify,by polymerase chain reaction (PCR),a 537bp fragment diagnostic for the presence of the transgene. A reverse transcriptase polymerase chain reaction (RT PCR) assay was designed to assess the relative proportion of transgene mRNA levels to that of the endogenous mouse tropoelastin mRNA. RESULTS A combination of rat tropoelastin cDNA and genomic DNA recombinants was used to generate three minigene constructs encoding the complete sequence of rat tropoelastin (tropoprom 1),the complete sequence lacking the domain encoded by exon 33(tropoprom 2),and the complete sequence lacking the domain encoded by exons 13~15(tropoprom 3). RT PCR amplification of total rat RNA produces a 516bp fragment and amplification of mouse RNA results in a similar sized 510bp fragment. CONCLUSION A series of transgenic mice harboring rat tropoelastin minigenes which constitutively express a particular isoforms are generated. This is direct evidence demonstrating the importance of tropoelastin isoforms for the normal assembly of elastin. OBJECTIVE To address the functional significance of tropoelastin isoforms, it was constructed of three rat tropoelastin minigenes that constitutively encode different tropoelastin isoforms, including those isoforms that lack the domains encoded by exon 33 and exons 13-15. METHODS The transgenes identified as tropoprom 1, tropoprom 2, and tropoprom 3 were created from rat tropoelastin genomic and cDNA clones. Oligonucleotide primers specific for the rat tropoelastin transgene (TRANS7 / 8 5 ’AGTCTCAACAGGTGCTGT 3’ and TRANS 16/17 5 ’AGTTCCAGCGCCTGTAAT 3’) were utilized to amplify, by polymerase chain reaction (PCR), a 537 bp fragment diagnostic for the presence of the transgene. A reverse transcriptase polymerase chain reaction (RT PCR) assay was designed to assess the relative proportion of transgene mRNA levels to that of the endogenous mouse tropoelastin mRNA. RESULTS A combination of rat tropoelastin cDNA and genomic DNA recombinants was used to generate thre e minigene constructs encoding the complete sequence of rat tropoelastin (tropoprom 1), the complete sequence lacking the domain encoded by exon 33 (tropoprom 2), and the complete sequence lacking the domain encoded by exons 13-15 (tropoprom 3). RT PCR amplification of total rat RNA produces a 516 bp fragment and amplification of mouse RNA results in a similar sized 510 bp fragment. CONCLUSION A series of transgenic mice harboring rat tropoelastin minigenes which constitutively express a particular isoforms are generated. This is direct evidence demonstrating the importance of tropoelastin isoforms for the normal assembly of elastin.
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