Recombinase-mediated mouse transgenesis by intracytoplasmic sperm injection

Area of Science:

• * Molecular Biology
• * Developmental Biology
• * Genetics

Background:

• * Traditional microinjection methods for animal transgenesis suffer from low efficiency due to poor embryo survival.
• * Existing intracytoplasmic sperm injection (ICSI)-based transgenesis techniques require improvement in efficiency and embryo viability.

Purpose of the Study:

• * To develop a novel bacterial recombinase-based transgenesis method with improved embryo survival and efficiency.
• * To investigate the efficacy of using E. coli RecA recombinase with single-stranded DNA (ssDNA) via ICSI in mouse oocytes.

Main Methods:

• * Developed a transgenesis method using bacterial recombinase RecA complexed with single-stranded DNA (ssDNA).
• * Employed intracytoplasmic sperm injection (ICSI) to deliver the RecA-ssDNA complex into mouse metaphase II (MII) oocytes.
• * Assessed RecA-dependent transgenesis and compared embryo survival rates with traditional methods.

Main Results:

• * Achieved RecA-dependent transgenesis in mouse oocytes using the novel ICSI-based method.
• * Demonstrated significantly improved embryo survival rates compared to pronuclear microinjection and previous ICSI approaches.
• * Showcased enhanced overall transgenesis efficiency due to better embryo survival.

Conclusions:

• * The novel bacterial recombinase (RecA)-based ICSI method offers superior embryo survival and transgenesis efficiency.
• * This approach provides a promising alternative to conventional microinjection techniques for animal transgenesis.
• * Opens new avenues for gene targeting via homologous recombination in mammalian pre-implantation embryos.