Especially designed for primary cells and very difficult to transfect
LAPA scoring of 7 day differentiating cells revealed colonies from the TET2-pCDNA3 condition having an average LAPA score of 0.9 compared to 0.45 for the control group, showing that the TET2-pCDNA3 transfect
colonies were on average 50% less differentiated than the control transfect
colonies at the time of analysis (Figure 3(a)).
Electroporation also improves the researcher's ability to transfect
for more difficult-to-transfect target cells, such as primary cells, and allows researchers to also transfect
entire tissues in vivo.
In the present study, a lentivirus-mediated VEGF-shRNA system was used for the following reasons: lentiviruses efficiently transfect
cells , lentiviral delivery of siRNA is suitable for local administration in ocular tissue, and the vector is transcribed in a DOX-dependent manner allowing easy control of shRNA transcription.
Calcium phosphate was used to transfect
the endophilin2-pEGFPC1 (Endo2-GFP) construct and its control, or endophilin2 siRNA (Endo2 siRNA) and its control, into the neurons.
Using lentiviral particles also allows effective direct visualization of Nuclei, Nuclear Membrane, Mitotic Chromosomes and Interphase Chromatin, Endosome, Endoplasmic Reticulum, Microtubule, Mitochondria, Golgi, Lysosome, Plasma Membrane, Cytoplasm and Peroxisome in hard to transfect
mammalian cells, stem cells and primary cells.
A key step in bringing this vector to the clinic was to demonstrate that it is both safe and feasible to transfect
the vector into human stem cells and for the RNA molecules to persist in the immune cells of the patient.
The generated recombinant virus was collected and transfected
into hFPPCs, followed by selection in 2 [micro]g/ml puromycin (Sigma) for 4 days.
SV40 large T antigen-transfected human leukemic Jurkat T cells (Jurkat-TAg) in logarithmic growth phase were transfected
with plasmid DNAs by electroporation using NEPA21 Super Electroporator (NEPAGENE, Japan).
Human CL 1-0 cells stably transfected
with PCNA expressing or control plasmids were treated with UV irradiation, H/A, and colcemid as described previously.
For MCU silencing, H9c2 cells were seeded at 2.5 x [10.sup.4] cells per well in 12-well plates and 24 h later were transfected
with 18.8,112.5, or 225 nM of siRNA-MCU designed or siRNA-Neg using the HiPerFect[TM] Transfection Reagent (Qiagen[R], Venlo, Netherlands), according to the manufacturer's protocols.