Analysis of DNA Integration in Transgenic Embryos

We have analyzed genomic DNA from transplantation-derived tadpoles to determine whether early integration of introduced plasmids into sperm or egg chromosomes is responsible for the nonmosaic expression observed (1). The pCARGFP plasmid was introduced into embryos by transplantation of sperm nuclei, and tadpoles 2.5 wk to 1 mo old were scored for nonmosaic expression of GFP in the somites; the presence and arrangement of pCARGFP were then determined by probing Southern blots of genomic DNA from these tadpoles with a 1-kb probe consisting of GFP sequences from one end of the linearized pCARGFP plasmid. We found that transplantation-derived tadpoles that did not express pCARGFP did not contain the plasmid, whereas each tadpole that expressed GFP contained between 5 and 35 copies of the plasmid/cell. The probe recognized four to eight bands in each GFP-expressing tadpole which were of unique sizes and were not found in other GFP-expressing tadpoles. These fragments represent putative junction points at which pCARGFP was integrated into the genome of each tadpole. Additionally, the probe recognized two common bands in all of the GFP-expressing tadpoles, corresponding to products formed by tandem and back-to-back concatemerization of the pCARGFP plasmid. By comparing the intensity of these bands relative to the putative junction fragments, we estimate that pCARGFP was integrated into the genome as single copies in some instances and as short (two to six copy) concatemers in other instances. Since the plasmid is expressed in all expected cells in the embryo, it is likely that most of these integrations occurred prior to the first cleavage division, assuring that all cells of the embryo would inherit several copies of the plasmid.

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