Microinjection as a method to increase the efficiency of isolation of primary colonies from inbred mice blastocysts in vitro
Abstract
We have estimated a perspective of the microinjection (MI) for isolation from blastocysts inbred mouse cells of the innercell mass (ICM) and trophoblast (TB) - sources of new stem cell lines of embryonic origin. It is shown that microinjection into the cavity of the blastocyst 10nl Witten’s medium and/or vital dyes (0.03% trypan blue and 0.1% phenol red) leads to damage of not more than 4-5% of the cells. In the process there are visible changes in the morphology of the blastocyst, which are almost completely leveled out during the first 24 h of incubation in the Witten’s medium. It was revealed that microinjection procedure doesn’t affect the subsequent development of the injected blastocyst to stage of primary colonies formation in vitro as a single population of interacting innercell mass and trophoblast. It’s also shown that the mechanical puncture of shiny shell (zona pellucida) and osmotic shock from the introduction of excessive amounts of fluid into the internal blastocyst cavity contribute to blastocyst outputting from zona pellucida and to colony formation (for NMRI and SHK cell lines up to 93 and 78 to 47 and 44% in control, respectively). Thus, microinjection small volumes of medium balanced by salt composition and/or solutions of vital dyes (less than 10 nl) can serve a sauxiliary method to increase the efficiency of isolation of primary colonies from inbred mice blastocysts with different genotype and to create new experimental models for biomedical research.
About the Authors
L. M. Mezhevikina
ФГБНУ «Центр экспериментальной эмбриологии и репродуктивных биотехнологий»
Russian Federation
Russian Federation
E. V. Khramtsova
ФГБУН «Институт биофизики клетки РАН»
Russian Federation
Russian Federation
T. I. Smolikhina
ФГБУН «Институт биофизики клетки РАН»
Russian Federation
Russian Federation
I. V. Kapralova
ФГБУН «Институт биофизики клетки РАН»
Russian Federation
Russian Federation
G. Yu. Kosovsky
ФГБНУ «Центр экспериментальной эмбриологии и репродуктивных биотехнологий»
Russian Federation
Russian Federation
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Review
For citations:
Mezhevikina L.M., Khramtsova E.V., Smolikhina T.I., Kapralova I.V., Kosovsky G.Yu. Microinjection as a method to increase the efficiency of isolation of primary colonies from inbred mice blastocysts in vitro. Journal Biomed. 2016;(1):25-36. (In Russ.)
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