Cloning Of The RRNA Genes And Construction Of The Multiple Locus Gene Targeting Vector Of Bos Taurus

The rRNA genes in animals are a kind of repetitive sequences with high GC content and complicated structure. The DNA sequences of rRNA repetitive gene family of Bos taurus were gained. The LA PCR method with LA PCR Taq DNA polymerase and GC buffer was used to amplify the complicated repetitive sequences of rRNA genes in Bos taurus after repeat experiment. Furthermore, the PCR products were cloned into pMD18-Simple T cloning plasmid. After sequencing identification the 3 rRNA genes and 2 internal transcribed spacers (ITS1 and ITS2) have been cloned. The results from sequencing showed that the lengths of five cloned fragments of rRNA gene were corresponded to 498bp, 2, 538bp, 1, 479bp, 2, 319bp and 3,326bp, respectively. The integrated contig with five sequences was identified to 8, 675bp, exactly, which separately comprised Bos taurus rRNA gene of 18S, ITS1, 5.8S, ITS2 and 28S with the length of l,873bp, 1, 072bp, 150bp, 1, 038bp and 4, 542bp. These above sequences were submitted to GenBank, and the corresponding accession numbers were granted to DQ222453. The measures for cloning the complicated DNA sequences such as the special rules of primer design, the improvement of reaction system, the selection of DNA polymerase and the adjustment of cycle parameter have been explored.The Bos taurus was the study object in this research. The vectors of multiple locus gene targeting on the repetitive internal transcribed spacers between rRNA genes in Bos taurus were constructed based on the BAC with Cre/loxP system. The key material of multiple locus gene targeting in vivo would be got. The plasmid of pYLSV-TDN with TK, HRDS2 and NEO genes has been constructed firstly. The TK-HRDS2-NEO DNA fragment digested from the plasmid of pYLSV-TDN by Not I/Hind III was inserted into the upstream...