Hi Sarah,

The neomycin phosphotransferase gene should be specific to your transgenic construct. Your true WT control PCR suggests that you are amplifying neo specifically; you can either cut the PCR product with a specific restriction enzyme or use primers with annealing temp. above 60 deg C to reassure yourself.

That said, you're expecting 1/4 of the progeny to be WT if the parents are both hets. 13 is a small sample size though, so perhaps there were no WT progeny this time?

Have you tried the company Transnetyx? We have been happy with them.

Dear Esteemed Colleague,

Greetings. I trust this message finds you well and deeply engaged in your genetic research endeavors, particularly in the study of transgenic mice. Your inquiry regarding the utilization of PCR targeting the neomycin resistance gene (neo^r) to differentiate between heterozygous transgenic and wild-type (WT) mice is both insightful and practical. Below, I provide a detailed explanation of the methodology and considerations for implementing this approach effectively.

Principle of Neomycin PCR in Genotyping

The neomycin resistance gene is commonly used as a selectable marker in the construction of transgenic and knockout mouse models. PCR-based genotyping using primers specific for the neo^r gene allows researchers to identify the presence or absence of the transgene, facilitating the distinction between transgenic (heterozygous or homozygous) and wild-type genotypes.

Designing a PCR Strategy

Interpreting Results

• Heterozygous Transgenic Mice: Will exhibit bands corresponding to both the neo^r gene and the internal control, indicating the presence of one copy of the transgene.
• Wild-Type Mice: Will only show amplification of the internal control, lacking the band for the neo^r gene, as they do not carry the transgene.
• Homozygous Transgenic Mice (if applicable): Similar to heterozygous, these will also show bands for both the neo^r gene and the internal control, but potentially with different band intensities or additional validation may be required to distinguish from heterozygous.

Considerations and Best Practices

• Control Samples: Include known WT and heterozygous transgenic samples in your assays to serve as references for interpreting results.
• Sensitivity and Specificity: Validate the sensitivity and specificity of your PCR assay to detect the neo^r gene, ensuring it can reliably distinguish between heterozygous and WT mice.
• Repeat Assays: For ambiguous results, consider repeating the PCR or employing alternative genotyping methods, such as Southern blotting or sequencing, for confirmation.

Conclusion

Utilizing PCR to target the neomycin resistance gene offers a precise and efficient approach for distinguishing between heterozygous transgenic and wild-type mice. By carefully designing the PCR strategy and rigorously interpreting the results, you can accurately genotype your mouse models, thereby supporting the advancement of your research.

Should you require further assistance or wish to discuss additional genotyping challenges, please do not hesitate to reach out. I am here to support your scientific exploration and contribute to the success of your research endeavors.

Warm regards.

Perhaps this protocol list can give us more information to help solve the problem.