Construction of a DNA Library Enriched with Mouse 4x Chromosome of T(X;4)37H Translocation: (flow cytometry/mouse DNA library/hybrid cell/chromosome translocation/X-inactivation).

ABSTRACT

Normal mouse chromosomes are routinely separated into only 5 peaks by the current flow cytometry. Since this limited resolution hindered isolation of the normal mouse X chromosome with an appropriate purity, we attempted to sort the mouse 4x chromosome, a larger translocation chromosome of T(X;4)37H, consisting of nearly the entire chromosome 4 and chromosome X by flow cytometry. To obtain a large number of cells having 4x chromosome for flow sorting, we isolated a somatic hybrid cell line MHH-1 formed between S194 myelome cell line and normal splenocytes from a male mouse carrying T(X;4)37H. Flow karyotyping of propidium iodide-stained chromosomes from MHH-1 cell line revealed an additional peak containing 4x chromosomes at about 80%. DNA purified from sorted 4x chromosomes was cloned into phage lambda gtWES after complete digestion with EcoRl restriction endonuclease. Thus a 4x chromosome-enriched library of about 4.4 × 104 recombinant phages was made and 13 single copy DNA clones specific to the X chromosome were isolated from the library so far.