Construction and characterization of a 10-fold genome equivalent rat P1-derived artificial chromosome library.

Journal article

A rat PAC library was constructed in the vector pPAC4 from genomic DNA isolated from female Brown Norway rats. This library consists of 215,409 clones arrayed in 614,384-well microtiter plates. An average insert size of 143 kb was estimated from 217 randomly isolated clones, thus representing approximately 10-fold genome coverage. This coverage provides a very high probability that the library contains a unique sequence in genome screening. Tests on randomly selected clones demonstrated that they are very stable, with only 4 of 130 clones showing restriction digest fragment alterations after 80 generations of serial growth. FISH analysis using 70 randomly chosen PACs revealed no significant chimeric clones. About 7% of the clones analyzed contained repetitive sequences related to centromeric regions that hybridized to some but not all centromeres. DNA plate pools and superpools were made, and high-density filters each containing an array of 8 plates in duplicate were prepared. Library screening on these superpools and appropriate filters with 10 single-locus rat markers revealed an average of 8 positive clones, in agreement with the estimated high genomic coverage of this library and representation of the rat genome. This library provides a new resource for rat genome analysis, in particular the identification of genes involved in models of multifactorial disease. The library and high-density filters are currently available to the scientific community.

Authors: Woon, P; Osoegawa, K; Kaisaki, P; Zhao, B; Catanese, J

• Publication status: Published
• Journal: Genomics
• Volume: 50
• Issue: 3
• Pages: 306-316
• Publication date: 1998-06-01
• DOI: 10.1006/geno.1998.5319
• EISSN: 1089-8646
• ISSN: 0888-7543
• Language: English
• Keywords: In Situ Hybridization, Fluorescence; Electrophoresis, Gel, Pulsed-Field; Polymerase Chain Reaction; Genetic Markers; Female; Rats; DNA Restriction Enzymes; Time Factors; Rats, Inbred BN; Trinucleotide Repeat Expansion; Chromosomes; Genomic Library; In Situ Hybridization; Cloning, Molecular; Animals; Chromosomes, Artificial, Yeast; Metaphase