Cellular Insertion

The third method of introducing genes into the germ line is a two-step process involving first the transfection of a transgene into embryonic stem (ES) cells, embryonic germ (EG) cells (also known as primordial germ cells), or fetal somatic cells during in vitro culture, and then the incorporation of the transgenic ES or EG cells into an inner-cell mass of an embryo or the insertion of the transgenic cell's nucleus into an enucleated oocyte by nuclear transfer (NT). The advantage of this procedure is that a particular genotype can be selected during in vitro culture before introduction of the cells into the embryo or NT. In addition, this technique provides the ability for site-specific insertion of a transgene by homologous recombination.

This approach with ES cells has been used extensively in the mouse but has not been effective in other mammalian animals because of extreme difficulty in isolating and maintaining ES or EG cells in the undifferentiated state during in vitro culture. Nuclear transfer (also known as animal cloning) is currently being extensively investigated in cattle, goats, and pigs. Consequently, cellular insertion by NT may become the method of choice for gene transfer in these species, because relatively few recipient hosts are required to produce transgenic founder animals.

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