GENETIC MODIFICATION OF RATS Russian patent published in 2019 - IPC C12N5/07 C12N5/10 C12N5/00 C12N5/02 

FIELD: biotechnology.

SUBSTANCE: invention refers to biotechnology, namely to creating a line of rat embryonic stem cells, obtaining a genetically modified rat and a composition for cultivating and maintaining pluripotency of rat embryonic stem cells. Method comprises culturing, which preserves pluripotency, in vitro of first layer of feeder cells, which are not modified for expression of leukemia inhibition factor (LIF), and a rat embryo at a morula or blastocyst stage, in which a glossy oocyte membrane is removed in a medium containing approximately 50 units/ml to about 150 units/ml of LIF, and a combination of inhibitors consisting of a MEK inhibitor and a GSK3 inhibitor. That is followed by transfer of growth of amorphous undifferentiated mass of rat embryonic stem cells to an in vitro cultivation well containing a second layer of feeder cells which are not modified for expression of LIF. That is followed by culturing growth under conditions including a medium containing from about 50 units/ml to about 150 units/ml of LIF, and a combination of inhibitors consisting of a MEK inhibitor and a GSK3 inhibitor, thereby maintaining pluripotency of the rat ES cells, and modifying the rat ES cell so that it contains a target genetic modification, wherein the rat ES cells are able to transmit the target genetic modification through the germline, do not express s-Mus, have a normal karyotype and form spheroid-like free-floating colonies in the culture. It is further possible to obtain a clone of the rat ES cell to obtain a genetically modified rat.

EFFECT: invention enables targeted genetic modification in rat embryonic stem cells that can be transmitted by genome.

47 cl, 21 dwg, 15 tbl, 7 ex