Hi Experts,
I am trying to transfect LUHMES cells and I've seen different protocols for transfection. Before or after differentiation, which one is better? And why?
I've tried transfecting the cells after differentiation, my experience was, they are difficult to transfect and got low efficiency. Why is that? I will transfect cells before differentiation, and hoping I would have better efficiency this time.
I'm just worried that DNA retention in the proliferating cells after transfection might also dwindle as differentiation requires at least 3 more days. What are your thoughts?
Thanks in advance for your help!
I have searched for the infection level (please see file; HepG2 fucoidan).
Fetal hepatocyte Hc has 5.0% virus among cell proteins.
Differentiated hepatoma HepG2 has 7.2% virus among cell proteins.
Healed hepatocyte HepG2 by fucoidan has 5.7% virus among cell proteins.
The virus content may be lower in developing cells or un-differentiated cells. Thus, ageing is only due to increase of infected virus.
Therefore, already residing conqueror virus in differetiated cells may treat the newcomer virus as a nuisance.
Thanks, Dr. Hayakawa. I understand what you mean. So there could be viral/plasmid DNA incompatibility that could lower the efficiency of the incoming DNA. Makes sense, however, how did you resolve that? (I will check the paper you sent). I appreciate your thoughts.
Further, I am now interested in Parkinson's disease (PD).
Then, I have searched for Dopamine-related proteins in my protein database, and infection of virus, which seems to rule the gene expressions.
Fetal hepatocyte Hc has no Dopamine-related proteins at all.
Hepatoma HepG2 has Dopamine D4 receptor at 2.8 μg/mg of cell protein, and also has Protein UL50 (HHV-5/Human cytomegallovirus/HCMV) at 2.7 μg/mg of cell protein.
Healed hepatocyte HepG2 (cultured with fucoidan) has Tyrosinase/Monophenol monooxygenase/LB24-AB/SK29-AB/Tumor rejection antigen AB at 0.26 μg/mg of cell protein, and also has Ribonucleotide reductase (HHV-5) at 1.9 μg/mg of cell protein.
HCC liver tissue (with PBC) has Tyrosinase/Monophenol monooxygenase/LB24-AB/SK29-AB/Tumor rejection antigen AB at 5.8 μg/mg of tissue protein, and also has 64 KD Lower matrix phosphoprotein (HHV-5) at 7.8 μg/mg of tissue protein.
Due thanks to Dr. Edward Alain Pajarillo (College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, FL, USA), this new finding suggests that the Parkinson's disease (PD) may be occurred by the infection of HHV-5.
I have previously estimated the cause of Dementia as follows; i.e.,
"I have further studied about the Dementia or Demenz in relation to virus. Dementia seems to be strongly linked to the infection or co-infection of Retrovirus, HAV, HCV, HHV-5, HHV-6, Coronavirus, Measles virus (MeV), Ebolavirus (EBOV), Togavirus, and Bacterio-phages of Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Haemophilus influenza. Haemophilus influenza uses nicotine (vitamin B3) and heme, and tobacco seems to be important to prevent the vitamin B3 deficiency and Dementia due to Haemophilus influenza."
There is also a question of nuclear envelope working as a barrier. Differentiated cells are usually not proliferating, so nuclear envelope is present all the time. If you use virus unable to transport its genetic material through nuclear pores, you will have a problem with transporting your transgene into nucleus, to be expressed or integrated with genome and then expressed. Usually oncoviruses are unable to transfect non-dividing cells, lentiviruses are able but if your lentivector has no cPPT sequence from pol gene, it will also be problematic. With dividing cells the level of modification is higher, as nucelar envelope is not present during mitosis. of course. Even with lentiviral vectors transfection of dividing cells gives much higher level of modified cells.
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