Construction of a DNA Library Enriched with Mouse 4

Area of Science:

• Cytogenetics
• Molecular Biology
• Genomics

Background:

• Standard flow cytometry resolution limits the purity of isolated normal mouse chromosomes, particularly the X chromosome.
• The translocation chromosome T(X;4)37H (4x chromosome) combines most of mouse chromosome 4 and the X chromosome.

Purpose of the Study:

• To enhance flow cytometry resolution for isolating the mouse 4x chromosome with high purity.
• To develop a strategy for obtaining sufficient 4x chromosomes for flow sorting and subsequent molecular analysis.

Main Methods:

• Established a somatic hybrid cell line (MHH-1) from a mouse carrying the T(X;4)37H translocation.
• Performed flow karyotyping on propidium iodide-stained chromosomes from the MHH-1 cell line.
• Cloned DNA from sorted 4x chromosomes into a phage lambda gtWES vector after EcoRl digestion.

Main Results:

• Flow karyotyping identified an additional peak representing 4x chromosomes in approximately 80% of MHH-1 cells.
• A 4x chromosome-enriched library containing approximately 4.4 × 10^4 recombinant phages was constructed.
• Thirteen single-copy DNA clones specific to the X chromosome were successfully isolated from the library.

Conclusions:

• The developed flow cytometry method enables the isolation of the mouse 4x chromosome with improved purity.
• The 4x chromosome-specific DNA library provides valuable resources for further genetic and molecular studies of the mouse X chromosome.