Genomewide two-generation screens for recessive mutations by ES cell mutagenesis.

Area of Science:

• Genetics
• Genomics
• Developmental Biology

Background:

• Forward genetic screens in mice typically use N-ethyl-N-nitrosourea (ENU) mutagenesis in male germlines.
• This traditional method requires three generations of breeding to achieve homozygous mutations for phenotyping.
• An alternative involves chemical mutagenesis of embryonic stem (ES) cells.

Purpose of the Study:

• To demonstrate the feasibility of performing genome-wide mutation screens in mice using mutagenized ES cells.
• To establish a more efficient strategy for generating homozygous mutations for genetic screens.
• To compare the efficiency of ES cell-based mutagenesis with traditional ENU mutagenesis.

Main Methods:

• Embryonic stem (ES) cells were treated with ethylmethane sulfonate (EMS).
• Chimeras were generated from mutagenized ES cells.
• Mice potentially homozygous for mutations were obtained by crossing chimeras to their daughters or by intercrossing chimera offspring.
• This resulted in a two-generation breeding scheme.

Main Results:

• The ES cell-based strategy successfully generated mice potentially homozygous for mutations in two generations.
• This method leverages the transmission of a single mutagenized diploid genome from chimeras.
• The strategy doubles the number of screenable mutations per pedigree compared to ENU mutagenesis.

Conclusions:

• Mutagenesis of ES cells offers a more efficient two-generation approach for mouse forward genetic screens.
• This method provides advantages in speed, mutation frequency, and flexibility compared to traditional ENU mutagenesis.
• The strategy facilitates the production of quality-controlled, long-term supplies of mutagenized cells for genetic screens.