Generation of genetically modified rats from embryonic stem cells

Area of Science:

• Stem Cell Biology
• Genetics
• Animal Models

Background:

• Generating genetically modified rats from embryonic stem (ES) cells has been challenging due to the lack of stable ES cells and effective injection methods.
• Previous attempts were hindered by the inability to reliably produce and maintain pluripotent rat ES cells suitable for genetic manipulation.

Purpose of the Study:

• To develop a reliable method for generating genetically modified rats using ES cells.
• To establish conditions for culturing authentic rat ES cells and enabling germline transmission.
• To create a monitoring system for rat ES cell pluripotency.

Main Methods:

• Generated Oct4-Venus transgenic (Tg) rats to monitor ES cell pluripotency.
• Defined optimal culture conditions using fetal bovine serum (FBS) and specific cell signaling inhibitors (for Rho-associated kinase, mitogen-activated protein kinase, TGF-beta, and glycogen synthase kinase-3).
• Utilized germline transmission of gene-manipulated ES cell clones for generating high-quality Tg rats.

Main Results:

• Established authentic rat ES cells expressing key markers (Oct4, Nanog, Sox2, Rex1) with a normal karyotype.
• Demonstrated successful embryoid body and teratoma formation from rat ES cells.
• Achieved germline transmission from all six ES cell lines across three rat strains, dependent on inhibitor presence during injection.

Conclusions:

• Developed a robust method for generating genetically modified rats from ES cells.
• Validated the utility of rat ES cells for gene targeting and transgenic manipulation.
• These genetically modified rats serve as valuable models for human disease research.