A PCR-based high-throughput screen with multiround sample pooling: application to somatic cell gene targeting.

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• Isolating rare gene-targeted clones in human somatic cells is challenging due to low transduction efficiencies.
• Existing methods may be time-consuming and inefficient for detecting low-frequency events.

Purpose of the Study:

• To develop and validate a systematic PCR screening method for efficient isolation of rare gene-targeted clones.
• To optimize the recovery of gene-targeted clones from human somatic cells transduced with targeting vectors.

Main Methods:

• Systematic PCR screening utilizing multiround sample pooling.
• Application to human somatic cells with 0.02-0.17% gene-targeted clones.
• Two rounds of pooling followed by de-pooling with successive PCR screening.
• Single-cell cloning for final isolation of gene-targeted clones.

Main Results:

• Successfully recovered rare gene-targeted clones from human somatic cells.
• The protocol demonstrated efficiency in identifying positive samples from pooled cell populations.
• The entire process can be completed within 4-8 weeks, depending on cell line proliferation.

Conclusions:

• The described method provides an effective strategy for isolating rare gene-targeted clones.
• This approach enhances the efficiency of genetic modification studies in human somatic cells.
• The protocol is adaptable and can be completed within a reasonable timeframe for molecular biology research.