An efficient i-GONAD method for creating and maintaining lethal mutant mice using an inversion balancer identified...

Area of Science:

• Genetics
• Molecular Biology
• Developmental Biology

Background:

• CRISPR/Cas gene editing is highly efficient, complicating the generation of homozygous lethal mutants.
• Maintaining heterozygous lethal alleles requires methods to suppress recombination.

Purpose of the Study:

• To develop an efficient in vivo method for generating and maintaining homozygous lethal mutants.
• To establish a Tprkb null knockout mouse strain with an embryonic lethal mutation.

Main Methods:

• Utilized improved genome editing via oviductal nucleic acid delivery (i-GONAD) for in vivo electroporation.
• Employed the B6.C3H-In(6)1J inversion to suppress recombination in lethal gene heterozygotes.
• Leveraged the Mitf coat color marker linked to the inversion for visual identification of mutations.

Main Results:

• Successfully generated the first Tprkb null knockout strain exhibiting embryonic lethality.
• Demonstrated that the B6.C3H-In(6)1J inversion effectively suppresses recombination.
• Confirmed visual recognition of the Tprkb mutation via the Mitf coat color marker.

Conclusions:

• The i-GONAD method combined with the B6.C3H-In(6)1J inversion provides an efficient strategy for creating and maintaining lethal mutants.
• This approach simplifies the study of embryonic lethal genes without requiring in vitro embryo manipulation.
• The developed method facilitates further research on the function of lethal genes.