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CHORI-243: Sheep (M) (Ovis aries) BAC Library |
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The CHORI-243 Ovine BAC library has been constructed by Michael Nefedov in Pieter de Jong's laboratory at BACPAC Resources, Children's Hospital Oakland Research Institute. The preparation of the library followed the cloning approach developed in our laboratory ( Osoegawa et al., 1998 ). Tim Smith (US Meat Animal Research Center (USMARC), at Clay Center in Nebraska provided blood from a ram of the Texel breed (animal # 200118011 (MARC population)). The particular animal has accumulated about 14% inbreeding over the last 5 generations of matings. The breed was selected because Texel is a popular terminal sire breed in several countries. In addition, the Texel breed served as the paternal grandsire breed of the sheep international mapping population. DNA was isolated from white blood cells by embedding the cells in agarose. Agarose embedded DNA was partially digested with a combination of EcoRI restriction enzyme and EcoRI Methylase and the fragments were size-fractionated by pulsed-field gel electrophoresis. DNA fragments from the appropriate size fraction were cloned into the pTARBAC2.1 vector between the EcoRI sites. The ligation products were then transformed into DH10B (T1 resistant) electro-competent cells (Invitrogen). The library has been arrayed into 384-well microtiter dishes and also gridded onto eleven 22x22cm nylon high-density filters for screening by probe hybridization. Each hybridization membrane represents over 18,000 distinct sheep BAC clones, stamped in duplicate. Library characterization was performed by Qing Cao, Teresa Ren and Kazutoyo Osoegawa. This work was funded in part by contributions from 5 scientific institutions:
Provisional data for CHORI-243 Ovine BAC Library:
Segment | 1 | 2 | All | Vector | pTARBAC2.1 | pTARBAC2.1 | pTARBAC2.1 | Restriction Enzyme | EcoRI/EcoRI Methylase | EcoRI/EcoRI Methylase | EcoRI/EcoRI Methylase | DNA Source | WBC | WBC | WBC | Plate Numbers | 1-240 | 241-528 | 1-528 | Plate Count | 240 | 288 | 528 | Empty Wells | 0 (0%) | 0 (0%) | 0 (0%) | Non-Recombinant Clones | 0 (0%) | 0 (0%) | 0 (0%) | Non-Insert Clones | N/A | N/A | N/A | Recombinant Clones | 92160 | 110592 | 202752 | Average Insert Size | 182 Kbp | 186 Kbp | 184 Kbp | Genomic Coverage | N/A | N/A | N/A |
Data on the CHORI-243 clone average insert size has been determined by Pulsed Field Gel Electrophoresis. Clone size distribution has been plotted graphically. While analyzing clones using pulse-field electrophoresis to determine the average insert size, non-insert clones containing a small deleted vector fragment consistent with sucrose resistance were observed. Further in depth characterization of the library is ongoing in our lab and data will be updated on our web page periodically.
Please direct questions concerning this library to either Pieter J. de Jong.
Ordering & Pricing information
The library is available in several formats. Individual clones, and high-density hybridization filter are obtainable. For ordering and shipping details, please view the ordering and pricing information ordering and pricing information page.
Academic and commercial users interested in a copy of the BAC library should contact Pieter J. de Jong ([email protected], fax: (510) 450-7924).
References:
1) Using comparative genomics to reorder the human genome sequence into a virtual sheep genome. Dalrymple BP, Kirkness EF, Nefedov M, McWilliam S, Ratnakumar A, Barris W, Zhao S, Shetty J, Maddox JF, O'Grady M, Nicholas F, Crawford AM, Smith T, de Jong PJ, McEwan J, Oddy VH, Cockett NE; International Sheep Genomics Consortium.
Genome Biol. 2007;8:R152
BACKGROUND: Is it possible to construct an accurate and detailed subgene-level map of a genome using bacterial artificial chromosome (BAC) end sequences, a sparse marker map, and the sequences of other genomes? RESULTS: A sheep BAC library, CHORI-243, was constructed and the BAC end sequences were determined and mapped with high sensitivity and low specificity onto the frameworks of the human, dog, and cow genomes. To maximize genome coverage, the coordinates of all BAC end sequence hits to the cow and dog genomes were also converted to the equivalent human genome coordinates. The 84,624 sheep BACs (about 5.4-fold genome coverage) with paired ends in the correct orientation (tail-to-tail) and spacing, combined with information from sheep BAC comparative genome contigs (CGCs) built separately on the dog and cow genomes, were used to construct 1,172 sheep BAC-CGCs, covering 91.2% of the human genome. CONCLUSION: We demonstrate that limited sequencing of BACs combined with positioning on a well assembled genome and integrating locations from other less well assembled genomes can yield extensive, detailed subgene-level maps of mammalian genomes, for which genomic resources are currently limited.
2) Chitko-McKown CG, Leonard JC, Moscatello D, Miller LC, Freking BA. Development of cell lines from the sheep used to construct the CHORI-243 Ovine BAC library. Anim Biotechnol. 2008;19:84-8
Two cell lines, designated MARC.OVSM, and MARC.OKF, have been generated by Carol Chitko-McKown at the U.S. Meat Animal Research Center, Clay Center, Nebraska. These cell lines were obtained from the aorta and kidney, respectively, from the same Texel ram used to create the CHORI-243 Ovine BAC library. The cell lines are publicly available from the NIA Aging Cell Repository at the Coriell Cell Respositories, Camden, NJ, USA.
Reference
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