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 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.
Wellcome Trust Centre For Human Genetics, University of Oxford, Headington, United Kingdom.
P Y
PY
Woon
K
K
Osoegawa
P J
PJ
Kaisaki
B
B
Zhao
J J
JJ
Catanese
D
D
Gauguier
R
R
Cox
E R
ER
Levy
G M
GM
Lathrop
A P
AP
Monaco
P J
PJ
de Jong
D000818:Animals; D002875:Chromosomes / Q000235:genetics*; D018244:Chromosomes, Artificial, Yeast; D003001:Cloning, Molecular / Q000379:methods*; D004262:DNA Restriction Enzymes; D016521:Electrophoresis, Gel, Pulsed-Field; D005260:Female; D005819:Genetic Markers; D015698:Genomic Library; D017403:In Situ Hybridization; D017404:In Situ Hybridization, Fluorescence; D008677:Metaphase; D016133:Polymerase Chain Reaction; D051381:Rats; D011914:Rats, Inbred BN; D013997:Time Factors; D019680:Trinucleotide Repeat Expansion
D005819:Genetic Markers; D004262:DNA Restriction Enzymes