GFP Expression in Mouse Fetuses Derived from Aggregation of Embryonic Stem Cells with Diploid or Tetraploid Embryos.

Abstract

Abstract:It is necessary to use a suitable marker gene to select embryos expressing transgene prior to transfer into recipient females especially in an efficient production of transgenic farm animals. Recently a green fluorescent protein (GFP) gene has been used as a marker gene for this purpose, because it is a self-fluorescent substance and does not require a substrate such as X-Gal in a lacZ gene. Using these characteristics of GFP, we transfected the GFP gene with the neomycinresistance gene under the control of the phosphoglycerase kinase promoter into mouse embryonic stem (ES) cells, and attempted to produce a transgenic mouse derived from aggregates of GFP-positive ES cells after the selection with G418 for 8 days with diploid or tetraploid embryos. There were some ES colonies expressing GFP at different levels after the selection with G418. These GFP-positive ES cells were used as a complete tight adhered clump for aggregation with diploid or tetraploid embryos and resulted in GFP expression in the inner cell mass (ICM) cells of aggregates, but no embryos expressing GFP in trophoblasts. At 9.5 days p.c., 6 out of 10 live fetuses (60%) for diploid⇔ES chimera, and all of 14 live fetuses (100%) for tetraploid⇔ ES chimera, in spite of head deformity, were transgenic fetuses. At 14.5 days p.c., among 7 normal embryos obtained from 21 embryos transferred to recipients, one fetus showed GFP expression in five organs (heart, kidney, liver, gonad and intestine), and 4 fetuses strongly expressed GFP in more than one organ as seen under the fluorescent dissecting microscope. These results indicate that enough transfected ES cells selected in a short time with G418 were able to be used for the selection of transgenic embryos prior to transfer to the recipient. Moreover, by using a complete tight adhered clump of ES cells for aggregation, the clump is able to completely contribute to a part of the ICM. The GFP gene may be useful and efficient as a marker especially for farm animals which have a long term pregnancy and a small litter size.

Further studies on thermal treatment of two‐cell stage embryos to produce complete embryonic stem‐cell‐derived mice by cell‐aggregation methods

“…Whether the weight gain is merely caused by the aggregation of two host embryos is not known. However, a similar phenomenon with unusual birthweight was found to occur in bovine aggregation chimeras (Iwasaki et al . 2000b).…”

“…A series of experiments were carried out as described previously by Iwasaki et al . (2000a) with minor changes in the number of ES cells.…”

“…Nevertheless, it has also been reported that in many cases transplanted embryos did not develop to term even if the ES cells employed for aggregation were in a state of early passage. Indeed, it has been reported that the putative tetraploid bovine embryos rendered by the electronic fusion method at the two‐cell stage, failed to develop as perfectly as murine tetraploid embryos (Iwasaki et al . 2000b).…”