Isolation of rat chromosome-specific paint probes by bivariate flow sorting followed by degenerate oligonucleotide primed-PCR

Abstract

The flow karyotype of rat chromosomes was determined by dual beam flow cytometry. Eighteen fractions were sorted and subsequently amplified by degenerate oligonucleotide primed-PCR as described by Telenius et al. (1992a, 1992b). The PCR products were labeled and used as probes for fluorescence in situ hybridization on rat fibroblast metaphases. The amplified chromosomes were detectable as bright chromosome paints and in most cases the signal was evenly distributed along the whole chromosome except for the centromeric region in half of the chromosomes. The results show that chromosomes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 19, 20, X and Y can be sorted as individual fractions, whereas chromosomes 11, 13, 14,15 and chromosomes 16, 17 and 18 are clustered together in the flow karyotype.

Reciprocal chromosome painting shows that genomic rearrangement between rat and mouse proceeds ten times faster than between humans and cats

“…The bivariate flow karyotype of the rat was more resolved than those previously reported (Hoebee et al, 1994;Shepel et al, 1995). We produced 18 single chromosome paints, when previous attempts produced only 13-15 single chromosome paints.…”

Genetic mapping of two blood pressure quantitative trait loci on rat chromosome 1.

“…Rat chromosome 1 DNA was obtained by flow sorting as described by Hoebee et al (17) and amplified by an initial PCR using a degenerate oligonucleotideprimer (DOP) (6-MW primer: 5Ј-CCGACTCGAGNNNNNNAT-GTGG-3Ј) as described by Telenius et al (18,19). Reamplification was carried out in a total vol of 100 l reaction mixture containing 5 l PCR products from the previous round of amplification, 1 ϫ PCR buffer, 200 M of each of the four dNTPs, 800 nM of 6-MW primer, and 1.25 U of Taq DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT).…”

Linkage Analysis and Construction of a Congenic Strain for a Blood Pressure QTL on Rat Chromosome 9

“…Table 1 gives markers from mouse chromosome 1 that are syntenic to rat chromosome 9 and which we found worked well in the rat. Table 1 also gives markers developed by us for rat chromosome 9 using DNA obtained from flow-sorted chromosomes using techniques described previously (Hoebee et al, 1994;Dukhanina et al, 1997;Deng et al, 1997a,b). The microsatellite marker for Ae3 (anionexchange protein 3, also called Slc4a3) was previously mapped to rat chromosome 9 .…”