Single-copy transgenic mice with chosen-site integration

Area of Science:

• Molecular Biology
• Genetics
• Transgenic Technology

Background:

• Comparing transgene expression is often complicated by variable copy numbers and random integration sites.
• Developing methods for controlled transgene insertion is crucial for accurate gene function studies.

Purpose of the Study:

• To establish a general method for introducing single-copy transgenes into specific mouse genome locations.
• To compare the expression driven by different promoters (chicken vs. human beta-actin) using a standardized transgene insertion approach.

Main Methods:

• Utilized homologous recombination in embryonic stem (ES) cells to achieve targeted integration of a single-copy bcl-2 transgene.
• Inserted the transgene 5' to the X-linked hypoxanthine phosphoribosyltransferase locus, enabling direct selection.
• Tested two promoters: chicken beta-actin and human beta-actin, driving murine bcl-2 cDNA.

Main Results:

• Homologous recombination generated mice with a single transgene copy at a defined genomic location.
• Transgene expression levels in ES cells were consistent for identical promoters but varied between different promoters.
• In contrast, random integration of the same promoter resulted in drastically variable bcl-2 expression.
• Both promoters directed broad transgene expression in mice derived from targeted ES cells.
• The human beta-actin promoter consistently showed higher expression than the chicken beta-actin promoter in vitro and in vivo.

Conclusions:

• Homologous recombination in ES cells provides a reliable method for generating genetically engineered mice with single-copy transgenes at specific loci.
• This approach eliminates confounding variables of copy number and insertion site, facilitating precise comparisons of promoter activity and transgene function.
• The human beta-actin promoter is a more potent driver of transgene expression compared to the chicken beta-actin promoter in this system.