BacMam Virus Transduced Cardiomyoblasts Can Be Used for Myocardial Transplantation Using AP-PEG-A Microcapsules: Molecular Cloning, Preparation, and In Vitro Analysis
Figure 3
Effect of NaBu and viral dose in H9c2 cells’ transgene expression. Cells were transduced with Bac-MGFP for 6 h in PBS at 27°C with and without 10 mM NaBu treatment at different viral doses. Quantification (fluorescein expression) of fluorescent in H9c2 cells (a) were made with relation to viral dosage 24 hpt. The average fluorescein-expressed (excitation 485 nm, emission 535 nm) values were normalized to that of the cells having 6 h of viral incubation in presence of PBS (taken as 100%) and are represented as normalized mean expression in percentage value. The data represent mean of three different readings. (b) Fluorescence images of transduced cells in absence and presence of NaBu are shown 24 hpt. (c) Detection of MGFP plasmid delivery in H9c2 cells 24 hpt in presence or absence of NaBu by standard PCR analysis. Cells were given equal viral dose with same viral incubation time. Nontransduced cells were taken as the control (mock) for the transduction procedure. The DNA was extracted from the transduced cells 24 h after transduction, and PCR analysis was done using primers that amplify the delivered MGFP gene. Amplification of housekeeping GAPDH gene serves as an internal control for the efficiency of polymerase reaction. Molecular weight (Mw) markers are low range DNA ladder. The similar band intensities in the agarose gel indicates that there was equal viral transduction in both +NaBu and −NaBu groups, although the corresponding protein expression was higher in +NaBu (as shown in (a) and (b)). This proves that NaBu does not interfere in viral transduction procedure but significantly improves the transgene expression.
