|
amaxa eNews #5
|
 |
|
|
Transfection of siRNAs into CML Primary Cells Using Nucleofection
|
Michaela Merkerova and Hana Bruchova
Institute of Hematology and Blood Transfusion, Department of Molecular Genetics, U nemocnice 1, 128 20 Prague 2, Czech Republic
|
 |
|
Abstract
|
|
RNA interference studies are based on transfection of short oligonucleotides (siRNAs) into cells and transfection efficiency is a crucial factor for such experiments. Most conventional transfection methods are suitable for immortalized cell lines, but are not efficient in primary cells. We performed comparative analysis of transfection methods for siRNA delivery into peripheral blood leukocytes from chronic myeloid leukemia (CML) patients. In conclusion, transfection by amaxa Nucleofector Technology represented the best and only suitable non-viral method for transfection of CML primary cells. Using Nucleofection we successfully transfected ~40% of the cells and achieved significant knock-down (~78%) of the target genes. Moreover, the cell viability was not affected as with other methods.
|
|
|
Introduction
|
|
RNA interference (RNAi) is the process of mRNA degradation induced by double-stranded RNA in a sequence-specific manner. In mammalian cultured cells, RNAi is typically induced by short-interfering RNA (siRNA) or a short hairpin RNA (shRNA) that induce targeted knock-down of gene expression, resulting in partial to full loss of function.
Basic requirements for successful RNAi experiments are effective siRNA sequence and efficient delivery method. For most immortalized cell lines, transfection with a lipid- or amine-based reagent yields satisfactory results. Electroporation is used for delivery into “hard-to-transfect” primary cells; however, it is often associated with decreased cell viability that is critical for further assays. This limitation can be bypassed using amaxa Nucleofector Technology that provides high transfection efficiency and does not affect cell viability.
Primary leukocytes belong to the category of “hard-to-transfect” cells. Although many genes associated with leukemias were examined by siRNA-mediated silencing in cell lines [1-4], their functional relevance in the disease should be confirmed in a more natural system, such as primary cells. In this study, we systematically tested a variety of non-vector based transfection methods (chemical transfection reagents, electroporation, Nucleofection) to determine optimal conditions for the delivery of siRNAs into peripheral blood leukocytes from chronic myeloid leukemia (CML) patients.
|
|
|
Material and Methods
|
|
CML cells
Total leukocytes were isolated from peripheral blood of 15 patients in chronic phase of CML at the time of diagnosis. K562 cell line (derived from a CML patient in blast crisis) was obtained from ATCC and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum in a humidified atmosphere at 37°C with 5% CO2.
Synthetic siRNA
Commercially available siRNAs against PCNA (#51014, Ambion), JNK2 (#1022632, Qiagen), and p38 (#sc-29433, Santa Cruz), and previously published siRNAs against MMP8 [5], MMP9 [6], and BCR-ABL [1] genes were used. A possible non-specific gene silencing effect was assessed using a scrambled control siRNA (#1022076, Qiagen).
TRANSFECTION METHODS
Chemical transfection
Metafectene [Biontex], Oligofectamine [Invitrogen] and siPORTAmine [Ambion] reagents were tested for chemical transfection. Transfections were performed in 12-well plates according to manufacturer protocols. Briefly, transfection reaction included optimized amount of siRNA (100pmol/sample), cells (1x106 cells), and a reagent (15ml of Metafectene, 4ml of Oligofectamine, or 5ml of siPORTAmine).
Electroporation
The cells (2x106) were electroporated in RPMI 1640 medium with 10% serum in 4mm cuvettes with 100pmol siRNA using Gene Pulser II instrument (BIO-RAD). Optimized parameters of electroporation were used (voltage 270V and capacitance 850mF). After electroporation, cells were incubated 5 min on ice and then replaced into the medium.
Nucleofection
The cells (5x106) were transfected with 100pmol siRNA using Cell Line Nucleofector Kit V (amaxa) according to manufacturer´s instruction. Electrical parameters of A-23, A-27, G-16, O-17, T-01, T-16, and T-20 on the amaxa Nucleofector Device were tested.
The transfection efficiency was monitored by fluorescent microscopy and flow cytometry to detect expression of pmaxGFP vector (amaxa). Cell viability was assessed by trypan blue exclusion method using a hemacytometer.
qRT-PCR
Transcript level of the silenced genes was measured after 48 hours post-transfection by quantitative real-time polymerase chain reaction (qRT-PCR) as described previously [5]. Relative gene expression was quantified on RotorGene 3000 instrument (Corbett Research) using calibration curve method and the data were normalized against GAPDH and b2-microglobulin.
|
|
|
Results
|
|
Optimization of Nucleofection conditions
Due to a predominant donor dependence of CML primary cells there is no single optimized Nucleofector protocol available for these cells. Therefore, it is necessary to test different Nucleofection settings to identify the most suitable Nucleofection condition with the highest transfection efficiency and cell viability.
Programs A-23, A-27, G-16, O-17, T-01, T-16, and T-20 were tested. For instance, program T-16 is recommended for K562 cells and it provides 74% of GFP-positive cells after pmaxGFP vector delivery in our hands (Figure 1A and 1B).
After transfection of the vector into CML primary leukocytes, fluorescence signal was observed in the cells transfected by programs O-17, T-16, and T-20. Efficiency of pmaxGFP transfection quantified by flow cytometry was as follows: program T-20: 36-42%, program T-16: 25-29%, program O-17: 14-14.5% (percentages of fluorescent cells). Based on these data, T-20 was assessed as the most efficient program (Figure 1A and 1C).
|
|
|
Figure 1: Transfection efficiency of Nucleofection. K562 cells (B) and freshly isolated CML primary cells (C) were transfected by pmaxGFP vector and cultured in a 12-well plate. After 24 hours, fluorescence signal was analyzed by microscopy (B, C) and flow cytometry (A).
|
Selection of optimal transfection method for CML primary cells
Previously, chemical reagents and electroporation have been successfully used for transfection of immortalized CML cell lines [1-4] and we examined their efficiency in peripheral blood leukocytes from CML patients. PCNA-siRNA or scrambled control siRNA were delivered into the cells using various methods.
Although chemical reagents provided high transfection efficiency in K562 cells [5], all of them (Metafectene, Oligofectamine and siPORTAmine reagents) failed to deliver siRNAs into the CML primary cells and we did not observed any significant knock-down of PCNA (Figure 2A).
Electroporation achieved higher transfection efficiency, but the electrical pulse markedly affected cell viability (~52% of viable cells). The gene expression of PCNA decreased only by ~37% (Figure 2B).
Using Nucleofection under optimal conditions, we successfully transfected siRNAs into CML leukocytes and achieved a significant knock-down of the target gene. After 48 hours of PCNA-siRNA delivery, the amount of PCNA transcript was lowered by 63%. Moreover, Nucleofection did not affect viability of transfected cells: 86% viable cells compared to 89% of the untreated control sample (Figure 2C).
|
|
|
Figure 2: Delivery of siRNAs into CML primary cells using various transfection methods. PCNA-siRNA and scrambled control siRNA (scr-siRNA) were transfected into CML primary cells by chemical transfection (A), electroporation (B), and Nucleofection (C). After 48 hours, level of PCNA mRNA was measured by qRT-PCR and cell viability was determined by trypan blue exclusion method.
|
Down-regulation of target genes by siRNA
To validate efficiency of Nucleofection and down-regulation effect, siRNAs against several well-known genes (PCNA, JNK2, p38, MMP8, MMP9, BCR-ABL) that are highly expressed in CML leukocytes, were selected. The siRNAs were transfected into CML primary leukocytes using program T-20 and relative transcript levels were measured after 48 hours of incubation (Figure 3). Expression of silenced genes decreased by 63-78% and cell viability was ~80% compared to 89% of the control sample.
|
|
|
Figure 3: siRNA-mediated silencing of the target genes. Gene specific siRNAs and scrambled control siRNA (scr-siRNA) were transfected by Nucleofection with program T-20 into CML primary cells. Relative gene expressions were measured by qRT-PCR after 48 hours post-transfection.
|
|
|
Conclusion and Outlook
|
|
amaxa Nucleofector Technology represents an innovative approach for transfection of primary cells. We performed comparative analysis of non-viral transfection methods for siRNA delivery into CML primary cells. Compared to others, Nucleofection provided the highest transfection efficiency without adversely affecting viability of CML leukocytes and turned out to be the best and only suitable non-viral transfection method for these primary cells. Using this technology, we successfully performed RNAi assays and achieved significant knock-down of different target genes with a high reproducibility.
Nucleofection can be applied not only to deliver siRNAs into primary blood cells, but it provides high efficiencies also for transfection of other types of nucleic acid molecules. Herein, we transfected 40% of cells by pmaxGFP vector and our preliminary data demonstrate that amaxa’s Nucleofector Technology is also a useful tool for miRNA delivery into K562-derived erythroid cells as well as erythroid progenitors from peripheral blood.
|
To top
|
|
References
|
|
[1] Sherr M, Battmer K, Winkler T, et al. (2003). Blood 101:1566-1568.
[2] Merkerova M, Bruchova H, Brdicka R. (2007). Leuk Res. 31:661-672.
[3] Merkerova M, Bruchova H, Kracmarova A, et al. (2007). Leuk. Lymphoma. 48:793-801.
[4] Merkerova M, Bruchova H, Brdicka R. (2007). Neoplasma 54:503-510.
[5] Merkerova M, Klamova H, Brdicka R, Bruchova H. (2007). Mol. Biol. Rep. 34:27-33.
[6] Sancéau J, Truchet S, Bauvois B (2003). J. Biological Chemistry 278:36537-36546.
|
|
|
|
| Invite a friend
| Recommend amaxa's eNews to a friend or colleague. |
|
| | | | |
|
|
The Nucleofector Technology, comprising Nucleofection Process, Nucleofector Device, Nucleofector Solutions, Nucleofector 96-well Shuttle System and Nucleocuvette plates and modules is covered by patent and/or patent pending rights owned by amaxa AG.
amaxa, Nucleofector, nucleofection, maxGFP, 96-well Shuttle and Nucleocuvette are either registered trademarks or trademarks of amaxa AG in the U.S. and/or Germany and/or other countries. Other product and company names mentioned herein are the trademarks of their respective owners.
amaxa disclaims all warranties, whether expressed or implied, including any warranty as to the quality, accuracy, safety, or suitability of the information provided in this e-newsletter for any particular purpose. Any use of the information contained on any page of this e-newsletter is evidence of agreement with these terms of use.
|
|
