Efficient, Non-Viral and Reproducible Protocol for Stable Knockdown of Genes in Mantle Cell Lymphoma Cell Lines
Authors: Hansen Simone Valentin , Blum Mia Koldby , Abildgaard Niels , Nyvold Charlotte Guldborg 
Published: November 2020

Introduction
The biological role of specific genes can be investigated using gene silencing by cell transfection with small interfering RNA (siRNA), which leads to degradation of the corresponding mRNA and reduced target protein expression. The study of biological functions in modified cell cultures often requires a long-lasting knockdown (KD) of gene expression, which can be obtained by viral delivery of short hairpin RNA (shRNA) or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 systems. However, these methods are laborious and require high level of laboratory safety. Hematopoietic cells, typically grown in suspension, are known to be difficult to transfect. Previous efforts to transfect lymphocytes using liposomes (H Guven et al, ExpHem2005) showed limited transfection efficiencies of only 10-30%. Transfection by electroporation leads to decreased viability, and therefore poses a risk of unintentional selection of cell subsets. Transfection of leukemia cells with siRNAs using Accell siRNA Delivery Media showed KD efficiencies of 56-65% and a viability of 57-95% (H Larsen et al,ExpHem2011). However, this was only investigated 24-72h post-transfection.
By whole transcriptome sequencing, we previously identified a number of candidate genes, includingLILRA4, PTPRJ and SYNE2, which were significantly upregulated in mantle cell lymphoma (MCL) cells compared to B cells from healthy donors (MH Hansen et al, ExpHem 2020). To investigate the biological function of these genes, an efficient and stable KD was required.
Aim
The aim of this study was to identify the most effective non-viral method for siRNA-mediated knockdown in MCL cells.
Methods
The MCL cell lines Mino, Jeko-1 and Granta 519, were transfected using three different methodologies; Lipofectamine 2000 (Invitrogen), Electroporation with Amaxa Nucleofector (Lonza), and Accell siRNA Delivery Media (Dharmacon). For Accell delivery a pool of 4 siRNAs targeting each gene was used (SMARTpool siRNA, Dharmacon). For the others, 2 pre-designed siRNAs (Ambion) targeting each gene were used. For all, a positive control siRNA targetingGAPDHwas included and different conditions tested e.g. siRNA concentration, incubation time and addition of serum. KD efficiency was evaluated by qPCR usingABLandGUSreference genes.
Viability of cells was measured either by flow cytometry staining with a live/dead marker or by trypan blue.
Optimization of the Accell protocol was performed in Granta 519 cells transfected with SMARTpool siRNAs targeting eitherSYNE2,LILRA4,orGAPDH. KD efficiency was evaluated with qPCR and viability was counted with trypan blue.
Results
Using Lipofectamine, at 24h a non-reproducible and non-efficient KD (&lt;40%) was achieved forLILRA4in the Jeko-1 cell line, while only 5-22% gene silencing was achieved for Mino. Using electroporation, a KD efficiency of 45-70% was achieved in Mino at 24h forGAPDH,LILRA4andPTPRJ, while inferior efficiency (&lt;15%) and viability were observed in Jeko-1 cells. However, in the Mino cell line,PTPRJwas almost fully re-expressed (80% expression) after 48h. Using transfection with Accell delivery medium,GAPDH,LILRA4,andPTPRJwere successfully knocked down in all tested cell lines with a median efficiency of 75% (range 50%-98%) after 72h. When performing KD in Accell delivery medium, the viability of the cells decreased after 72h and dropped to 26-39% at day 5 and 6, and a decrease was found in the total number of cells, suggesting that proliferation may also be affected. By adding 10% fetal bovine serum (FBS) to this medium after 48h, the viability was kept above 66.7% for up to 6 days, and the total number of cells was comparable to that observed in normal culture medium, while the KD efficiency was still preserved. In Ganta 519 cells with addition of FBS, an average KD efficiency of 67% was achieved forLILRA4(range 56-72%) at 72h-5 days, while the average KD efficiency ofSYNE2was 77% (range: 67-95%) at 72h-5 days.
Conclusion
This study propose that transfection with SMARTpool siRNAs in Accell Delivery Medium with addition of 10% FBS 48h post transfection is an applicable procedure for siRNA-mediated KD in MCL suspension cell lines. This protocol permits efficient, non-viral, and stable KD across different MCL cell lines without affecting the cells' ability to proliferate and survive, and is therefore suitable for investigating biological processes.

Disclosures
No relevant conflicts of interest to declare.