Construction And Characterization Of Bacterial Artificial Chromosome (BAC) Library Of Chinese Ujumqin Sheep And Study Of IP-10 Gene

This work was aimed to seek an efficient approach for the conserving of important domestic animals in imminent danger or on the edge of extinction. A bacterial artificial chromosome (BAC) library of the nationally protected Chinese Ujumqin sheep was successfully established using the Hind III site of the vector pCC1BAC, comprising of 153,600 clones arrayed in 400 384-well microplates, with an average insert size of 90-100 kb and approximately fivefold coverage of the sheep genome, which yielded a theoretical probability of 99.4% of isolating a particular DNA sequence. The percentage of non-insert clones was 3.2%, false positive ratio was 0.86%. BAC clones of the library were stable in the bacterial host for at least 100 passages. These results suggested that the Chinese Ujumqin sheep BAC library provided a high-quality pool for conserving the nationally protected breed resource at genomic level for long term storage.In order to effectively using the Chinese Ujumqin sheep bacterial artificial chromosome library, in this study, we using Qpix multi-functional automatic mechanical arm to combine and mix the BAC clones to establish the Ujumqin sheep bacterial artificial chromosome library PCR screening system. The PCR screening system is divided into bacteria library and DNA library, which including bacteria and DNA of 40 super-pools, 400 plate pools, 16 row pools and 10 column pools , respectively. 26 pairs of microsatellite marker primers including BM6465 covering sheep chromosome were screened the Ujumqin sheep bacterial artificial chromosome library, showed each of these markers were positive in the library, the positive clone number were 3 to 9, average number was 5.1, which was consistent with fivefold coverage of the sheep genome.A Ujumqin sheep ear marginal tissue (USEM) fibroblast line, containing 147 cryovials with 4×10~6 cell each, was successfully established from 33 Ujumqin sheep ear marginal tissues using explant culture and cryopreservation techniques. The cell line met all the criteria from the American Type Culture Collection (ATCC). Not only has the germline of this important sheep breed been preserved at the cell level, but also valuable material had been provided for genome, postgenome and somacloning research. Moreover, the establishment of this technical platform may provide both technical and theoretical support for storing the genetic resources of other animals and poultry at the cell level.In order to verify the quality of Chinese Ujumqin sheep bacterial artificial chromosome library, in this study, the the super pool, pool row, column and plate pools pool were PCR screened by interferon inducible protein-10 gene was, the results indicated there were 6 positive superpools and eight positive clones were obtained, USB0501F18/041, USB0501L18/041, USB1209A8/119, USB1405P10/135, USB2201C3/211, USB2206C3/216, USB2503D17/243 and USB2902G8/282, respectively. The average insert size of positive clones was about 90kb. The fragments of PCR product was about 322bp bands. The sequencing report of recombinant pGEM-T-IP-10 indicated that the result was sheep IP-10 gene sequences. The eukaryotic expression plasmid pEGFP-N3-IP-10 was digested with Xhol I and BamH I indicated an visible fragment of 309 bp and 4.7 kb vector band. Recombinant expression vector pEGFP-N3-IP-10 was highly expressed in Ujumqin sheep fibroblast cells, the green fluorescence was uniformly distributed in the cells, and the target cells were proliferated and divided. pEGFP-N3-IP-10 and the control group of no insert pEGFP-N3 vector both expressed in target cells, the expression efficiencies were highest 48h after transfection (P <0.05).