Quantitative estimation of chimerism in mice using microsatellite markers

Area of Science:

• Developmental Biology
• Genetics

Background:

• Embryonic stem (ES) cell technology is crucial for generating genetically modified organisms.
• Assessing the degree of chimerism is essential for validating ES cell contribution, especially for germline transmission.

Purpose of the Study:

• To establish a quantitative method for assessing chimerism using polymorphic microsatellite markers.
• To evaluate the utility of microsatellite analysis for determining the percentage of ES cell contribution in chimeric mice.

Main Methods:

• Generation of chimeric mice from SV129 ES cells injected into OF1 blastocysts.
• Utilized polymerase chain reaction (PCR) to amplify polymorphic microsatellite markers (CKMM system).
• Developed linear calibration models to correlate DNA ratios with PCR signal ratios for quantitative analysis.

Main Results:

• Successfully generated chimeric mice, with 3 out of 13 males proving to be germline chimeras.
• Demonstrated that microsatellite markers accurately quantify chimerism across various tissues.
• Achieved high correlation (≥0.98) between DNA and signal ratios for known chimerism levels.
• Prediction accuracy for % SV129 DNA ranged from 2-17% difference across repeated measurements.

Conclusions:

• Polymorphic microsatellite markers provide a robust and quantitative method for assessing chimerism.
• This technique is valuable for monitoring ES cell contribution, particularly in cases where coat color or biochemical markers are insufficient.
• The method holds significant potential for applications in interspecies ES cell research.