Generation of chicken Z-chromosome painting probes by microdissection for screening large-insert genomic libraries

Abstract

A strategy for rapid generation of chicken sex chromosome-Z painting probes has been developed using microdissection. Whole chromosome painting probes (WCPs) were prepared from 10–15 copies of mitotic metaphase chicken Z chromosomes. The microisolated chromosomes were subjected to PEG/proteinase K treatment in a collection drop to release DNA, which was then amplified using a degenerate oligonucleotide-primed shuttle PCR (DOP-Shuttle-PCR) strategy. Size distributions of the PCR products were analyzed by agarose gel electrophoresis and smears of DNA were revealed that ranged in size from 200–800 bp, without any evidence of preferential amplification. Both specificity and complexity of the probes have been analyzed by Southern blot and fluorescence in situ hybridization (FISH). Non-specific hybridization was efficiently blocked by using chicken competitor DNA. Analysis of the WCPs produced shows that collectively they provide uniform hybridization signals along the entire length of the chicken Z chromosome. To demonstrate one possible application of these complex probes, we screened a large-insert bacterial artificial chromosome (BAC) chicken genomic library to select Z chromosome-specific clones. To address specificity of the selected clones and to physically map them to the Z chromosome, FISH analysis was used. Of the 3 clones initially tested, one clone (C3) carrying a 250-kb insert mapped to the distal portion of the short arm of the chicken Z chromosome. Therefore, this technique has provided appropriate probes for screening large-insert genomic libraries. Further application of these probes includes the analysis of chromosome rearrangements, studies of cases of heteroploidy involving the Z chromosome, positional cloning of Z-linked genes and studies on mechanisms of sex-chromosome evolution in birds.

Micro- and macrochromosome paints generated by flow cytometry and microdissection: tools for mapping the chicken genome

“…It is essential that PCR techniques are sensitive enough to amplify only a single chromosome, as most are indistinguishable under the microscope, and, hence, it is not practicable to locate the same chromosome on a different metaphase. This represents a significant advance in the field of chicken gene mapping, as, in previous studies, microdissected paints were made from a template of several isolated chromosomes (e.g., Ponce de Leó n, 1996; Zimmer et al, 1997). In other words, although chromosome paints generated by microdissection of larger chicken chromosomes are reported in the literature, this study is the first, to our knowledge, to report the generation of chromosome paints from single microdissected microchromosomes.…”

“…Chicken/ human cross-species chromosome painting to establish interspecific synteny between these two extensively mapped vertebrates may ultimately become a reality. Furthermore, unlabeled paints represent libraries from which chromosome-specific sequences can be isolated (e.g., Ponce de Leó n, 1996;Zimmer et al, 1997).…”

Testing probiotic strain Escherichia coli Nissle 1917 (Mutaflor) for its ability to reduce carriage of multidrug-resistant E. coli by elderly residents in long-term care facilities

“…Ten colonies were picked randomly from each primary E. coli (without norfloxacin) plate culture prepared from urine or faeces during the treatment and follow-up periods of the trial. The subcultures were stored in 96-well microtitre plates at 220 uC as described by Zimmer et al (1997). To identify E. coli Nissle 1917 among the stored isolates, bacterial cells were transferred from a storage well by toothpick to PCR tubes.…”

Chromosomal rearrangements in�cattle and�pigs revealed by�chromosome microdissection and�chromosome painting

“…[33,41]). Painting probes have been produced by microdissection, for instance in cattle, pigs and horses [3,13,24], as well as chickens [14,16,46], but mainly for comparative Zoo-FISH purposes. In this paper, we report the use of conventional microdissection for the production of whole chromosome and chromosome arm painting probes, and their use for the subsequent accurate characterisation of two distinct chromosomal rearrangements: (1) a pericentric inversion of chromosome 4 in a boar, and (2) a mosaic with 15% of the cells bearing a 2/5 translocation in a bull.…”