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 Home > Resources > Libraries
  CHORI-250 Rhesus macaque BAC Library

  

The CHORI-250 Rhesus macaque BAC library has been constructed at the Children’s Hospital Oakland Research Institute, BACPAC Resources, by Dr. Baoli Zhu using the cloning techniques developed in our laboratory (Osoegawa et al., 1998).  Approximately 40 ml blood was collected by Marta L. Marthas (mlmarthas@ucdavis.edu) at the California Regional Primate Research Center (CRPRC) and was extracted at CHORI for the preparation of high molecular weight DNA embedded in agarose.  To prepare segment 1 of the library, the DNA was partially digested with a combination of EcoRI restriction enzyme and EcoRI methylase followed by double size fractionation using CHEF gels. Size fractionated DNA was cloned into the pTARBAC2.1 vector between the EcoRI sites. For segment 2, the DNA was partially digested with MboI followed by double size fractionation. The size fractionated DNA was cloned into the pTARBAC1.3 vector between the BamHI sites. The ligation products were transformed into DH10B (T1 resistant) electrocompetent cells (BRL Life Technologies). The library has been arrayed into 384-well microtiter dishes and gridded onto 12 22x22cm nylon high-density filter for screening by probe hybridization. Each hybridization membrane represents over 18,000 distinct rat BAC clones, stamped in duplicate. BAC library construction was funded by a grant from NIH (HG01165-06).

Information with respect to DNA source of Rhesus macaque: Local ID: MMU 25311 (A*01 and B*01 positive), male, 10 years old, healthy (10 kg), assigned to an indoor breeding colony operated under a CRPRC SPF production grant (J. Roberts, PI). The Rhesus blood donor is of Indian origin and was born at the CRPRC.

Provisional data for R. Macaque BAC library

Segment
DNA
Restriction Enzyme
Plate Numbers
Total Plates
Empty Wells
Empty Wells (%)
1
pTARBAC2.1
Adult Male Leukocyte
EcoRI
1-288
288
2,163
2.0
2
pTARBAC1.3
Adult Male Leukocyte
MboI
289-576
288
1,271
1.1
Total Library
 
 
 
1-576
576
3,434
1.5

 

Segment
Non-insert Clones (%)
Non-insert clones
Recombinant Clones
Insert size (Average)
Redundancy (Genome size:
3.0 Mb)
1
2.8
Approx. 3,036
125
Approx.105,268
171 Kbp
6.0
2
1.8
Approx. 1,968
77
Approx. 107,276
155 Kbp
5.5
Total Library
2.3
Approx. 5004
202
Approx. 212,544
163 Kbp 
11.5

click here for a legend of the previous tables.

Data on the CHORI-250 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 on going in our lab and data will be updated on our web page periodically.

A list of CHORI-250 non-recombinant clones is also available. The library is being used at the Baylor College of Medicine effort to assemble the rhesus macaque genome. Low-redundancy shotgun sequencing is done from pools of BAC clones ("row and column pools"). The derived sequences are being used to associate sequences with specific BACs and correlate these sequences & BACs with specific genomic regions of another species: the human genome. The derived BAC sequences are being used in combination with sequence data derived by the whole-genome shotgun sequencing (WGSS) effort at BCM. The combination of WGSS data and the BAC-pool derived data results in "eBAC" sequences. Please note that the WGSS sequences are being obtained from a different female indian macaque obtained by BCM from the San Antonio Regional Non-Human Primate Center. The pooled shotgun sequencing method and its implementation to map rhesus BACs relative to the human genome, has been described by Milosavljevic et al. 2005. To downlaod a PDF file of this manuscript, please follow this link:    http://www.genome.org/cgi/reprint/15/2/292.

BAC clones mapped by "Pooled genomic indexing" of the rhesus macaque BAC library can be found on the Baylor website (www.genboree.org).

Please direct questions concerning this library to either Pieter J. de Jong or Kazutoyo Osoegawa .

 
  Ordering & Pricing information 

The library is available in several formats. Individual clones, and high-density hybridization filters are obtainable. For ordering and shipping details, please view the ordering and pricing information page.

Academic and commercial users interested in a copy of the BAC library should contact Pieter J. de Jong ( pdejong@chori.org ), fax: (510) 450-7924).

 
  Reference

 

Aleksandar Milosavljevic, Ronald A. Harris, Erica J. Sodergren, Andrew R. Jackson, Ken J. Kalafus, Anne Hodgson, Andrew Cree, Weilie Dai, Miklos Csuros, Baoli Zhu, Pieter J. de Jong, George M. Weinstock, and Richard A. Gibbs. Pooled genomic indexing of rhesus macaque. Genome Res. 2005 February; 15(2): 292-301.

 

 

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