1999 Volume 18 Issue 1 Pages 119-123
We examined possible solvents for DNA which can be directly introduced into the nuclei of cultured cells by physical microinjection through glass capillaries. DNA of the plasmid pCH110containing lac-Z, a β-galactosidase gene, or the plasmid pSV2neo containing ned, a neomycinphosphotransferase gene, suspended in solvents commonly used in the fields of cell biology and biochemistry, such as PBS, TBS, or distilled water alone, was microinjected into the nuclei of NRK cells. Transient expression of the lac-Z gene and induction of neomycin-resistance occurred almost at the same level for all these solvents, except for EDTA-containing solutions, e. g., TE. For TE solution, the transient and stable expression levels were reduced, and could be recovered by supplying magnesium ions. Furthermore, we examined the effects of EDTA on the macromolecule barrier of nuclear pores. Following EDTA injection, fluorescein-conjugated albumin, that does not ordinarily pass through nuclear pores freely, immediately leaked from the nucleus of injected cells into the cytoplasm. These results indicate that microinjection of EDTA into a nucleus disrupts nuclear pore function, possibly resulting in inhibition of transient and stable expression of introduced genes.
