Differential expression and stability of foreign genes introduced into human fibroblasts by nuclear versus cytoplasmic microinjection.
We have compared cytoplasmic- and nuclear-delivered, glass needle-mediated microinjection protocols for their ability to support both transient and stable phenotypic expression of reporter gene constructs in non-immortalized human skin fibroblasts cultures. Microinjection of form I (covalently closed circular, supercoiled) plasmid pMC38 DNA into the nucleus of human cells resulted in high levels of transiently expressed p110gag-myc oncoprotein as detected by immunofluorescence microscopy. Likewise, the nuclear delivery of a plasmid construct bearing the entire simian virus 40 genome induced the formation of morphologically transformed foci in approximately 6% of the recipient cell population. In contrast, the introduction of plasmid DNA by the cytoplasmic route proved virtually incapable of supporting either transient gene expression or morphological transformation. In situ autoradiography of cells injected with 3H-labelled plasmid DNA revealed that whereas the material delivered directly into the nucleus was retained by this subcellular compartment for prolonged times (greater than or equal to 48 h), the radiolabelled DNA molecules introduced via the cytoplasmic route did not reach the nucleus and appeared to be substantially degraded within 8 h following injection. These results indicate unequivocally that nuclear injection is the route of choice when monitoring foreign gene expression in human cells.
Department of Medicine, Cross Cancer Institute, Edmonton, Alb, Canada.
R
R
Mirzayans
R A
RA
Aubin
M C
MC
Paterson
D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D003593:Cytoplasm / Q000378:metabolism*; D005347:Fibroblasts; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D008845:Microinjections; D016274:Oncogene Protein p55(v-myc) / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D013539:Simian virus 40 / Q000235:genetics; D014170:Transformation, Genetic / Q000235:genetics
D016274:Oncogene Protein p55(v-myc)