Team:TECHNION-ISRAEL/hpc7
Experiments in HPC-7
HPC-7 cells were chosen as our second model cell line because they display many characteristics of hematopoietic stem cells.
When we initially began our project, we strived to prove our system works in difficult to transfect HPC-7 cells. These cells are notoriously difficult to work with and require a tremendous amount of care and diligence to culture. HPC-7 cells are not commonly used in research, and there is very limited experimental data available for them. As a result, we had to use trial and error in order to determine even the most basic protocols.
We began by attempting to culture the cells, using slightly different conditions and mediums, until we found what worked best. The next challenge was transfecting the cells. For that purpose, we tried four different commercial chemical reagents: lipofectamine2000, LT1, polyJet, and TransIT2020. The transfection efficiencies were very low so we decided to conduct an optimization experiment with the most promising regents: lipofectamine2000 and TransIT2020. Based on a previous experiment conducted in human hematopoietic cell lines [1] .we checked several transfection parameters: cell concentrations, DNA:Reagent ratios, and reagent exposure time.
Additionally we tried electro-transfection methods since many papers described promising results with these kinds of cells [2] [3] .
We contacted various companies in an attempt to acquire a suitable electroporator. After a month, our attempts bore fruit and we got our hands on the NEPA21 electroporator, generously supplied by Almog Diagnostics. Unfortunately, the electroporation process was too aggressive for these sensitive cells. We managed to achieve modest transfection rates, but almost all of the cells died as a result of the electroporation process.
Accepting failure in the electroporation experiments was very difficult for us, especially after all the time and effort we had invested. Still determined, we pressed on, attempting once more to transfect these difficult cells with chemical reagents, but with the powerful EF-1a promoter, known for its high expression in stem cells [4] . We conducted the experiment once again using lipofectamine2000 and TransIT2020 and transfected an Ef1a-GFP reporter plasmid to measure our efficiency.
In Figure 1 you can see that the transfection was finally successful, and we got relatively high transfection efficiency.
Figure 1: Fold change of the transfection with lipofectamine 2000 and transit 2020 in HPC-7.
Conclusion :
Transfection of HPC-7 cells is not straightforward procedure and requires many tissue-culture skills. In our experience, the best transfection results obtained using chemical reagents, especially lipofectamine 2000 in a DNA:Reagent ratio of 1:3.
- Floch, Virginie, et al. "Cationic phosphonolipids as non viral vectors for DNA transfection in hematopoietic cell lines and CD34+ cells." Blood Cells, Molecules, and Diseases 23.1 (1997): 69-87.
- Volpe, Giacomo, et al. "Regulation of the Flt3 gene in haematopoietic stem and early progenitor cells." PloS one 10.9 (2015): e0138257.
- Dasse, E., et al. "Distinct regulation of c-myb gene expression by HoxA9, Meis1 and Pbx proteins in normal hematopoietic progenitors and transformed myeloid cells." Blood cancer journal 2.6 (2012): e76.
- Mikkola, Hanna, et al. "Lentivirus gene transfer in murine hematopoietic progenitor cells is compromised by a delay in proviral integration and results in transduction mosaicism and heterogeneous gene expression in progeny cells." Journal of virology 74.24 (2000): 11911-11918.
Department of Biotechnology & Food Engineering
Technion – Israel Institute of Technology
Haifa 32000, Israel
igem.technion.2017@gmail.com
