CONSTRUCTION AND CHARACTERIZATION OF A 10-FOLD GENOME EQUIVALENT RAT P1-DERIVED ARTIFICIAL CHROMOSOME LIBRARY

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 inser t size of 143 kb was estimated from 217 randomly isolated clones, thus representing approximately 10-fold genome coverage. This coverage pro vides a very high probability that the library contains a unique seque nce in genome screening. Tests on randomly selected clones demonstrate d that they are very stable, with only 4 of 130 clones showing restric tion 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 n ot all centromeres. DNA plate pools and superpools were made, and high -density filters each containing an array of 8 plates in duplicate wer e prepared. Library screening on these superpools and appropriate filt ers 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 identificatio n of genes involved in models of multifactorial disease. The library a nd high-density filters are currently available to the scientific comm unity. (C) 1998 Academic Press.