Lentil root protoplasts: a transient expression system suitable for coelectroporation of monoclonal antibodies and plasmid molecules.
Protoplasts were isolated from lentil (Lens culinaris) roots and their suitability as a transient expression system was investigated. After transfecting the protoplasts with the beta-glucuronidase (GUS) gene by either electroporation or polyethylene glycol (PEG), the specific activity of the reporter enzyme and the cell viability were determined. Electroporation was more effective than PEG treatment as transfection procedure and its efficiency was affected by the plasmid length. The feasibility of electro-transferring at the same time (coelectroporation) inhibitory anti-lipoxygenase monoclonal antibodies and the GUS-carrying plasmid pBI 221 was investigated as well. The amount of transferred immunoglobulins was quantitated by ELISA and the inhibitory ability of monoclonal antibodies on the intracellular target enzyme was determined. Evidence is presented for the successful coelectroporation of immunoglobulins and plasmid DNA into lentil protoplasts, the two types of macromolecules acting independently of each other in the recipient cells.
Maccarrone
M
M
Bijvoet Center for Biomolecular Research, Department of Bio-Organic Chemistry, Utrecht University, The Netherlands.
Veldink
G A
GA
Finazzi Agrò
A
A
Vliegenthart
J F
JF
D000911:Antibodies, Monoclonal; D018274:Electroporation; D007887:Fabaceae / Q000166:cytology / Q000235:genetics*; D015870:Gene Expression; D017930:Genes, Reporter; D005966:Glucuronidase / Q000096:biosynthesis / Q000235:genetics; D008084:Lipoxygenase / Q000235:genetics; D018517:Plant Roots / Q000166:cytology / Q000235:genetics; D010946:Plants, Medicinal; D010957:Plasmids / Q000235:genetics*; D011523:Protoplasts; D014162:Transfection / Q000379:methods*
D000911:Antibodies, Monoclonal; D008084:Lipoxygenase; D005966:Glucuronidase