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RPCI-71: Zebrafish (Danio rerio) BAC Library

The RPCI-71 Zebrafish BAC Library was constructed by Aaron Mammoser and Kazutoyo Osoegawa in Pieter de Jong's Laboratory using a locally-developed cloning approach (Osoegawa et al., 1998). Genomic DNA was isolated from 7,000 embryos obtained from the Max Planck Institute in Tubingen,Germany (see below about strain). The agarose-embedded DNA was partially digested with a mixture of EcoRI and EcoRI Methylase. Size-purified DNA fragments were cloned into the pTARBAC2 vector between the EcoRI sites with simultaneous replacement of the pUC-link stuffer fragment. The ligation products were transformed into DH10B electro-competent cells (BRL Life Technologies).  The BAC clones have been arrayed into 384-well microtiter dishes. The library can be screened by hybridization with probes using 22x22cm nylon high density filters each representing colonies derived from 48 microtiter dishes of the library. Library construction was supported by a sub-contract from a grant awarded to Dr. Robert Geisler at The Max-Planck-Institut fuer Entwicklungsbiologie. This library is one of several libraries used for the international zebrafish genome sequencing project (see: http://www.sanger.ac.uk/Projects/D_rerio/library_details.shtml). Many clones have also been used for genome mapping using a BAC fingerprinting approach.

The clones can be found in various public databases using either the "internal" Sanger nomenclature or the "external" NCBI recommended nomenclature. For example, CH71-15B7 (NCBI nomenclature) corresponds to a clone from the CHORI-71 BAC library found in microtiter dish (384-well type) #15, at the intersection of row "15 and column "7". The same clone in the "internal" Sanger nomenclature is labeled as: "bZ15B7". When ordering clones from BACPAC Resources Center (BPRC, @CHORI) using the online ordering system, please use the NCBI nomenclature.

Overviews of the BAC and fosmid libraries used for the zebrafish sequencing and mapping projects can be found at the Sanger "Fingerprinting web page" and the Sanger "Zebrafish Libraries" page.

The name of the zebrafish strain used for the BACs is "Tue" (Tuebingen). The characteristics of this fish line are described in the following paper: Rauch, G.-J., Granato, M. and Haffter, P. (1997). A polymorphic zebrafish line for genetic mapping using SSLPs on high-percentage agarose gels. Technical Tips Online T01208.

We prefer NOT to distribute this particular library because it does not satisfy our high quality standards. We are, however, distributing individual clones and providing screening filters. The lower quality of the library is based on the smaller average inserts (much less than the 150 kb minimal standard) due to a fraction of clones with rather small inserts. In addition, a minor fraction of the clones contain bacterial sequences instead of the zebra fish genomic DNA. The contamination resulted most likely from bacterial growth in the original collection of embryos used for DNA extraction. The library was nevertheless useful for the fingerprinting and contig assembly of many genuine zebra fish BACs. Hence, various BAC derived from this library have been completely sequenced and the derived sequences have been entered and annotated in the EBI and NCBI databases. We are committed to the distribution of individual BACs annotated in the international sequence databases.

click here for a legend of the previous tables.

Data on the RPCI-71 clone average insert size has been determined by Pulsed Field Gel Electrophoresis. Clone size distribution has been plotted graphically. The average insert size determined by pulsed field analysis of 182 random clones is 138 kb. This average is in disagreement with the average insert size obtained by the mapping group by counting up the individual HindIII restriction fragments (85 kb average reported).

Copies of this BAC library are not available, but high-density colony filters can be obtained (see Filter Availability). Individual clones identified after screening can be obtained as stab cultures at a cost of $80 per clone in addition to a $35 set-up charge per shipment. Shipping will be done by Federal Express and charged to the recipients account number.

Please visit the ordering information page if you want to place an order of high-density filters and individual clones. Please contact BACPAC Resources ([email protected]).