A genome-wide, end-sequenced 129Sv BAC library resource for targeting vector construction

Area of Science:

• Genomics
• Molecular Biology
• Mouse Genetics

Background:

• Gene targeting in mice commonly uses 129Sv-derived embryonic stem (ES) cells due to their germline colonization efficiency.
• Gene targeting relies on homologous recombination, influenced by vector characteristics and target locus.
• Efficient gene targeting is crucial for creating mouse models for research.

Purpose of the Study:

• To develop a comprehensive bacterial artificial chromosome (BAC) library from 129S7 ES cell DNA.
• To facilitate the rapid construction of gene-targeting vectors for mouse research.
• To improve the efficiency of gene targeting in 129-derived ES cell lines.

Main Methods:

• Generated and performed double-end sequencing of 84,507 BACs from AB2.2 ES cell DNA (129S7/SvEvBrd-Hprtb-m2).
• Aligned BAC sequences to the mouse genome and visualized on the Ensembl genome browser.
• Utilized recombineering for constructing targeting vectors from the BAC library.

Main Results:

• The BAC library provides an average insert size of 110.68 kb.
• Achieved 3.63-fold and 1.24-fold genome coverage across autosomes and sex chromosomes, respectively.
• Covered over 97% of the mouse genome and 99.1% of Ensembl genes.
• Demonstrated efficient gene targeting in 129-derived ES cells using recombineered DNA from the BAC library.

Conclusions:

• The developed BAC library is a valuable public resource for mouse genomics and gene targeting.
• This resource enables rapid construction of targeting vectors, enhancing gene modification efficiency.
• Facilitates the creation of precise mouse models for studying gene function and disease.