The electroporation-mediated DNA transfer and expression of the gene encoding ß-glucuronidase (GUS) in the protoplasts and partially digested cells of a marine green alga Ulva lactuca was studied. In order to obtain protoplasts, vegetative blades of freshly collected plants were digested with a mixture of 1.5% Cellulysin and 1.5% abalone acetone powder for 3 h. Protoplasts were separated from partially digested thalli by sieving and centrifugation. The GUS reporter gene carried on the plasmid pBI121 was introduced into both isolated protoplasts and partially digested cells by electroporation in the presence of polyethylene glycol (PEG-8000). Different parameters of electroporation were varied in order to achieve 50% viability of the protoplasts. Histochemical assays revealed a transient expression of GUS activity in both electroporated protoplasts and partially digested cells 2 to 7 days after electroporation, indicating that an exogenous gene can be introduced into the protoplasm with or without the removal of the cell wall. Subsequent culture led to the regeneration of protoplasts into filamentous or disc-like multicellular plantlets. However, none of these regenerated plants stained positively for GUS activity.Brought to you by |
1. It has previously been demonstrated that metabolism of drugs via a single enzymatic pathway, particularly CYP3A4, is associated with increased risk for drug-drug interactions (DDI). Quantitative experimental systems as well as integrated prediction models to assess such risk during the preclinical phase are highly warranted. 2. The present study was designed to systematically investigate the performance of human cryopreserved hepatocytes in suspension to predict fraction metabolized via CYP3A (fmCYP3A) by assessing the ketoconazole sensitive intrinsic clearance (CLint) for five prototypical CYP3A substrates with varying degree of CYP3A dependent CLint in twelve individual hepatocyte batches. 3. We demonstrate that in contrast to well predicted mean hepatic metabolic clearance (CLH) and mean fmCYP3A data, the variability in CYP3A contribution for compounds having multiple metabolic pathways cannot be predicted from inhibition experiments using ketoconazole as inhibitor. Instead, data in the present paper indicate that the variability is larger after inhibition of CYP3A for compounds having multiple metabolic pathways. 4. It is therefore recommended to estimate the average CLint and fmCYP3A for a given test compound in a series (n = 10) of individual human hepatocyte batches.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.