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Construction and Characterization of Large-Insert Genomic Libraries (BAC and Fosmid) from the Ascidian Botryllus schlosseri and Initial Physical Mapping of a Histocompatibility Locus

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Abstract

The colonial protochordate Botryllus schlosseri is genetically manipulable and represents a potential model organism for a variety of biological disciplines, including immunology, stem cell biology and development. This article presents the construction and characterization of both BAC and fosmid genomic libraries of the 725-Mbp B. schlosseri genome. The BAC library currently consists of 2× genome coverage with an average insert size of 80 kb. The fosmid library is at 11× genome coverage with an average insert of 40 kb. B. schlosseri is a small organism containing a large number of compounds that hinder DNA purification. Thus a number of protocols had to be modified in order to make purified, high molecular weight inserts for cloning, including both gel purification and insert concentration techniques. Both libraries were characterized by using them in initial physical mapping of a single histocompatibility locus, and were found to be representative and functional. These libraries are important tools for physical mapping and positional cloning in the B. schlosseri genome, and the techniques adapted to make them are suitable for use on other organisms in which high molecular weight DNA is difficult to purify.

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Acknowledgements

The authors are indebted to Tatsuya Ota, Jerry Spangrude, Tom Miyake, and particularly Chris Amemiya for many discussions and helpful tips. The Botryllus hand picking team included Karla Palmeri, Justin Conneau, and Diana Laird. Diana Laird is also thanked for critical reading and suggestions for the manuscript. We also thank Kathi Ishizuka, Karla Palmeri, and Vicki Takacs for Botryllus collection and mariculture. This work was supported by National Institutes of Health grant INRSA AI10332 to A.W.D. and R01-AI41588 to I.L.W.

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  1. Department of Pathology, Stanford University School of Medicine, Stanford CA 94305, and Hopkins Marine Station, Pacific Grove, CA 93950, USA

    Anthony W. De Tomaso & Irving L. Weissman

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  1. Anthony W. De Tomaso
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  2. Irving L. Weissman
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Correspondence to Anthony W. De Tomaso.

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De Tomaso, A.W., Weissman, I.L. Construction and Characterization of Large-Insert Genomic Libraries (BAC and Fosmid) from the Ascidian Botryllus schlosseri and Initial Physical Mapping of a Histocompatibility Locus . Mar. Biotechnol. 5, 103–115 (2003). https://doi.org/10.1007/s10126-002-0071-4

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  • DOI: https://doi.org/10.1007/s10126-002-0071-4

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