Microdissection and microcloning of the mouse X chromosome.

Abstract

A wild mouse (CD) karyotype in which all the chromosomes bar the X, 19, and Y, are fused as metacentrics has been used for the microdissection and microcloning of a specific mouse X chromosome region. Dissection of a proximal region of the X chromosome encompassing the genetic loci Hprt to Tfm and including mdx has yielded 650 clones. A number of the recovered clones containing sizable inserts have been confirmed as X chromosome specific. This X chromosome bank ofclones provides a start point for the isolation ofthe mdx locus.It is now clear that microdissection and microcloning can be applied to all mouse chromosomes, including the X chromosome, yielding premapped banks of clones that will greatly aid in the isolation and characterization of important genetic loci.The mouse X chromosome has become a paradigm for the study of a variety of biological phenomena of molecular interest: X chromosome inactivation (1, 2), sex determination (3-6), and the comparative evolution of mammalian chromosomes (7,8 methanol/acetic acid (3:1) and spread by air-drying. Metaphase spreads were stained with 1% Giemsa for photography.Microdissection and Microcloning. Unstained metaphase spreads of the CD strain were prepared on coverslips according to the methods described above. Coverslips were inverted over an oil chamber and chromosomes were dissected by using glass microneedles with the aid of a de Fonbrunne micromanipulator as described (11,12). All manipulations were observed under a phase-contrast microscope with a long working distance. Specially fine microneedles were drawn on a de Fonbrunne microforge and were used for the microdissection of the proximal region of X chromosomes well separated in the metaphase plate. After dissection, DNA was prepared and cloned essentially according to published micromethods (11, 12) but with some modifications. Briefly, fragments were pooled in a drop of proteinase K/0.5% NaDodSO4 residing on a separate coverslip in the oil chamber. After dissection was completed, the oil chamber was incubated at 37TC for 2 hr. After incubation, the nanoliter drop was extracted several times with phenol. After chloroform extraction to remove residual phenol the nanoliter drop was transferred to a fresh oil chamber and a nanoliter drop of restriction enzyme (EcoRI, 50 units/hd) was added and digestion proceeded by incubation at 370C for 2 hr. Phenol extraction to inactivate enzyme and chloroform treatment were repeated with subsequent transferance of the microdrop to a fresh oil chamber. A nanoliter microdrop ('400 pg) of EcoRI-digested vector, XgtlO, was added, followed by a nanoliter microdrop (0.4 unit) of ligase. Ligation proceeded overnight at 4°C. After ligation, the microdrop was taken up in several microliters of buffer for in vitro packaging. Recombinants were selected by plating on an Escherichia coli C600hflA-host (14, 15). Individual plaques were picked and plate stocks were prepared by propagation on SM32 (16) using standard procedures.Analysis of Microclones. A plate lysate method was u...

Microdissection of and microcloning from the short arm of human chromosome 2.

“…As in previous microdissection and cloning experiments with D. melanogaster and mammalian DNA (8,23,24), the size range of inserts is much smaller than that observed for total genomic libraries (15). This has been attributed to acid hydrolysis of the DNA during fixation of the chromosomes (8 Al of the single-copy probes were labeled with (a-32P)dCTP and hybridized to the panel filters.…”

“…This has been attributed to acid hydrolysis of the DNA during fixation of the chromosomes (8 Al of the single-copy probes were labeled with (a-32P)dCTP and hybridized to the panel filters. All clones were found to map to the short arm of chromosome 2; the data for two of these, pHM18 and pHM76, are shown in Fig.…”

“…This method was first applied to DNA from individual bands of Drosophila * Corresponding author. melanogaster polytene chromosomes (24) and was extended to mammalian chromosomes with the isolation of clones mapping to the mouse t complex (23) and to a proximal region of the mouse X chromosome (8). In mouse genetics, the technique has fulfilled the promise of fine genetic and molecular mapping of individual chromosome regions and allows the isolation and characterization of previously unclonable genetic loci.…”

“…The microcloning procedures were essentially as previously described (8,23,24). All solutions involved in the purification and cloning steps were pipetted with siliconized glass micropipettes; the volume added was equal to the size of the collection drop at each stage.…”

The Development of Chromosome Microdissection and Microcloning Technique and its Applications in Genomic Research

“…One of the major concerns for the successful application of chromosome microdissection and microcloning technique was the extensive depurination of the chromosomal DNA caused by acid treatment during the sample fixation in chromosome preparation [14]. At present, depurination had been reduced or avoided by improving the chromosome preparation procedure [27,28].…”