Efficient modification of the APRT gene by FLP/FRT site-specific targeting

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• The FLP/FRT system is a powerful tool for site-specific recombination.
• Its application in CHO cells for gene targeting requires further characterization.

Purpose of the Study:

• To establish and characterize the FLP/FRT system at the APRT gene in CHO cells.
• To evaluate its efficiency in gene targeting and intrachromosomal recombination.
• To demonstrate its utility in constructing chromosomal substrates for recombination studies.

Main Methods:

• Establishing the FLP/FRT system in CHO cells targeting the APRT gene.
• Analyzing gene targeting frequencies with and without FLP stimulation.
• Utilizing Southern blotting to characterize resulting cell lines.
• Employing screening for additional markers to enrich for integrants.
• Testing intrachromosomal recombination by reexposing integrants to FLP.

Main Results:

• FLP stimulation increased targeting frequencies 6-22 fold (1-5 X 10(-5)).
• Analysis of 52 APRT+ cell lines showed 56% FLP-targeted integrants, 33% convertants, and 11% undefined patterns.
• Enrichment strategies yielded 95% integrants.
• Intrachromosomal recombination (popouts) was stimulated over 200-fold without affecting adjacent homologous recombination.
• The system successfully generated chromosomal substrates for homologous and illegitimate recombination.

Conclusions:

• The FLP/FRT system is effective for site-specific recombination in CHO cells.
• It significantly enhances gene targeting efficiency.
• The system provides a versatile platform for constructing specific chromosomal substrates for diverse genetic studies.