|
Modifications to our previously introduced system for laser microbeam cell surgery were carried out in the
present work to match animal cells.
These modifications included: 1- Using other laser system that used before, Excimer laser with 193 and 308 nm
wavelengths. The used laser here, is He-Cd with low power and 441.5 nm wavelength in the visible region. 2- Instead of
using pulsed laser, we used here CW He-Cd chopped by electrical chopper, which is synchronized with the mechanical
motion of the mobile stage with step 40 microns, according to cell dimensions to avoid puncturing the same cell twice.
The advantages of the modified here laser setup for gene transfer is: it is less damaging to the sensitive animal cell which
has thin cell membrane.
The present work aimed to: 1- Design a modified laser microbeam cell surgery, applicable to animal cells, such
as fibroblast cells 2- To examine the efficiency of such system.
3- To assure gene transfer and its expression in the used
cells. 4- To evaluate the ultra damages produced from using the laser beam as a modality for gene transfer.
On the other wards, to introduce: safe, efficient and less damaging modality for gene transfer in animal cells.
To achieve these goals, we applied the introduced here home-made laser setup with its synchronized
parameters to introduce pBK-CMV phagemid, containing LacZ and neomycin resistance (neor )genes into BHK-21
fibroblast cell line.
The results of the present work showed that: 1- Our modified laser microbeam cell surgery setup proved to be
useful and efficient tool for gene transfer into fibroblast cells.
2- The presence and expression of LacZ gene was
achieved using histochemical LacZ assay. 3- Selection of G418 antibiotic sensitivity assay confirmed the presence and
expression towards stability of neor gene with time. 4- Presence of LacZ and neor genes in the genomic DNA of
transfected fibroblast cells was indicated using PCR analysis.
5- Transmission electron microscopy indicated that, no
ultradamages or changes for cell; membrane, organilles or any component of transfected fibroblast cell as a result of
using laser microbeam compared with control cell.
|
