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Construction of a DNA Library Enriched with Mouse 4

Ichiro Takahashi1, Takayuki Ueda2, Yosuke Kameoka1

  • 1Department of Virology and Rickettsiology, National Institute of Health, Kamiosaki, Shinagawa-ku, Tokyo 141, Japan.

Development, Growth & Differentiation
|June 7, 2023
PubMed
Summary
This summary is machine-generated.

Researchers improved flow cytometry to isolate the mouse 4x chromosome, aiding X chromosome research. This technique enhances chromosome sorting for genetic studies.

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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.