Gene introduction into mouse blastocysts via “pricking”

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Abstract
It is a well-known phenomenon that cultured mammalian cells that have been pricked in the presence of foreign DNA can be transformed. This micromanipulation ‘pricking’ technique was applied to mouse blastocysts to determine whether uptake of exogenous DNA would occur in the embryos. The middle region of the inner cell mass (ICM) was pricked three times in each blastocyst in a medium containing a linearized plasmid DNA. When the 60 treated blastocysts were transferred to the uterine horns of pseudopregnant females, 30 developing fetuses (50%) at the mid-gestation stage were obtained. Twenty-two of the 30 fetuses (73%) had less than 1 copy of the foreign DNA per diploid cell, as revealed by polymerase chain reaction (PCR)-Southern analysis, a sensitive technique combined with Southern blot processing of the PCR products. The 8 other fetuses were negative for the foreign DNA. When blastocysts were pricked in the presence of vector DNA coupling E. coli β-galactosidase (β-gal) gene to a mouse metallothionein-I (MT-I) promoter and assessed for β-gal activity histochemically after 1 and 5 days of culture in the presence of 1 μM CdCI2, at least 65% of the embryos exhibited β-gal activity mainly in the ICM region. These results indicate that mouse blastocysts can be transfected with a relatively high efficiency after pricking, and that the introduced gene expression occurs. This approach provides a means of mapping the regulatory elements of genes that are active in the mouse blastocyst ICM, and may be useful in investigating the fate of the ICM cells in an intact blastocyst by labeling them via pricking technique.