Microinjection of DNA into the nuclei of human vascular smooth muscle cells.
BACKGROUND
It is challenging to successfully transfect human vascular cells by conventional techniques. We evaluated the efficiency of transfection of human smooth muscle cells (SMC) using a method of direct nuclear microinjection of DNA constructs.


METHODS
The nuclei of explanted human saphenous vein SMC were microinjected with the plasmid pCMVbeta, containing the lacZ gene for beta-galactosidase (beta-gal). Efficiency of injection and expression were assessed by histochemical staining for beta-gal. Injected SMC were subjected to standard assays of viability and migration.


RESULTS
Parameters affecting the conditions of injection were systematically analyzed to achieve optimal transfection efficiency. A vertical injection resulted in a twofold increase in expression of beta-gal compared to a horizontal approach. A DNA concentration of 100 ng/microl (390 copies/injection) provided a maximal rate of expression. No further increase in expression was evident at higher concentrations. Maximal expression was achieved with a time of injection of 200-500 ms, an injection pressure of 5-10 psi, and a pipette tip size of 0.6 microm, resulting in an injection volume of 0.03 pl. Cytoplasmic injection did not result in gene expression. The ability of SMC to migrate under videomicroscopy was not altered by the injection process. Optimizing all injection parameters resulted in cell viability >95% and efficiency of injection of 59%.


CONCLUSIONS
DNA encoding a variety of intracellular proteins can be efficiently microinjected into human vascular SMC. Coupled with the use of videomicroscopy, this technique can allow for the evaluation of genes that might modulate important cellular processes such as proliferation and migration.
Division of Vascular Surgery, University of Massachusetts Medical School, Worchester, MA 01655, USA.
Peter R
PR
Nelson
K Craig
KC
Kent
D002465:Cell Movement; D002467:Cell Nucleus; D002470:Cell Survival; D002478:Cells, Cultured; D003470:Culture Media; D003593:Cytoplasm; D004247:DNA / Q000494:pharmacology*; D015870:Gene Expression; D006801:Humans; D007763:Lac Operon; D008845:Microinjections; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000502:physiology*; D011312:Pressure; D014162:Transfection / Q000295:instrumentation / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics
D003470:Culture Media; D004247:DNA; D001616:beta-Galactosidase