PRECEDENTES VINCULANTES VERSUS PRINCIPIO DE INDEPENDENCIA Y DE

hTPC2 cells was due to the over-expression of hTPC2, sequence-specific short hairpin RNA (shRNA) was used in order to knockdown / knockout hTPC2 protein expression. 4 plasmids encoding anti-hTPC2 shRNA and an mRFP reporter were designed and prepared by my collaborators, Professor Jianjie Ma and Dr. Zui Pan.

RNA interference (RNAi) is a form of gene regulation that occurs post- transcriptionally. It was first observed in 1998 when double stranded RNA (dsRNA) selectively abolished the twitching phenotype of Caenorhabditis elegans when injected directly into the body (Fire et al., 1998). This led the way for investigations involving the silencing of functional genes by exogenous application of dsRNA. RNAi can be achieved by incorporating DNA encoding shRNA into an expression vector. When this sequence is expressed, the resultant RNA consists of a sense and anti-sense strand which, due to its own complementarity, associates with itself by forming a hairpin structure that is able to interact with both the target mRNA and the cell machinery capable of mediating RNAi.

The 4 shRNA probes used in these experiments were targeted to specific sequences of hTPC2 (Fig. 4.4, Table 4.1) and were annealed to the BamH1 and EcoRI restriction sites of the pSIREN-DNR plasmid (Clontech, CA, USA). Each probe contained a sequence encoding a mRFP reporter protein. The presence of the mRFP reporter allowed for the visual determination of successful shRNA transfection. To maximise the success of shRNA mediated knockdown/knockout of hTPC2, the 4 probes were mixed in equal proportions and treated as a single solution for subsequent

experiments. It is important to note that the process of transfection of a plasmid into the cells could adversely affect protein expression/synthesis in cells. Thus, in order to discern whether the knockdown of hTPC2 was due to the shRNA rather than the transfection process, a control plasmid containing scrambled shRNA was also used in parallel to the test plasmids (Table 4.1). The effect of shRNA-mediated knockout / knockdown of hTPC2 expression in HA-hTPC2 cells was characterised using Ca2+ imaging. However, the ability of shRNA against hTPC2 to reduce hTPC2 expression was first verified using both Western blot and immunocytochemical labelling of intracellular proteins. When verifying the shRNA by Western blot, the level of β-

actin in each sample was used as a further control. If the transfection process does not adversely affect protein expression / synthesis one would expect, therefore, the level of β-actin in shRNA-transfected cells to be similar to non-transfected cells.

MAEPQAESEP LLGGARGGGG DWPAGLTTYR SIQVGPGAAA RWDLCIDQAV VFIEDAIQYR 60 SINHRVDASS MWLYRRYYSN VCQRTLSFTI FLILFLAFIE TPSSLTSTAD VRYRAAPWEP 120 PCGLTESVEV LCLLVFAADL SVKGYLFGWA HFQKNLWLLG YLVVLVVSLV DWTVSLSLVC 180 HEPLRIRRLL RPFFLLQNSS MMKKTLKCIR WSLPEMASVG LLLAIHLCLF TMFGMLLFAG 240 GKQDDGQDRE RLTYFQNLPE SLTSLLVLLT TANNPDVMIP AYSKNRAYAI FFIVFTVIGS 300 LFLMNLLTAI IYSQFRGYLM KSLQTSLFRR RLGTRAAFEV LSSMVGEGGA FPQAVGVKPQ 360 NLLQVLQKVQ LDSSHKQAMM EKVRSYGSVL LSAEEFQKLF NELDRSVVKE HPPRPEYQSP 420 FLQSAQFLFG HYYFDYLGNL IALANLVSIC VFLVLDADVL PAERDDFILG ILNCVFIVYY 480 LLEMLLKVFA LGLRGYLSYP SNVFDGLLTV VLLVLEISTL AVYRLPHPGW RPEMVGLLSL 540 WDMTRMLNML IVFRFLRIIP SMKPMAVVAS TVLGLVQNMR AFGGILVVVY YVFAIIGINL 600 FRGVIVALPG NSSLAPANGS APCGSFEQLE YWANNFDDFA AALVTLWNLM VVNNWQVFLD 660 AYRRYSGPWS KIYFVLWWLV SSVIWVNLFL ALILENFLHK WDPRSHLQPL AGTPEATYQM 720

TVELLFRDIL EEPEEDELTE RLSQHPHLWL CR 752

Fig. 4.4. Amino acid sequence for human two-pore channel subtype 2

The amino acid sequence of human TPC2 (hTPC2). The predicted transmembrane and P-loop regions are highlighted in yellow and cyan respectively. The 4 amino acid sequences corresponding to the mRNA sequences against which the 4 shRNA probes are targeted, are indicated by red italics.

Name of Probe Nucleotide number Nucleotide sequence Si-hTPC2 A 216-238 GCTTTACCGACGGTATTACTCGA Si-hTPC2 B 585-608 GCTGCAGAACTCCTCTATGATGAA Si-hTPC2 C 923-945 GCAGCCATCATCTACAGTCAGTTC Si-hTPC2 D 1556-1578 GCTCTGGCTGTGTACCGATTGCCA Control GATCCGTGCGCTGCTGGTGCCA- ACTTCAAGAGATTTTTTGCTAGCG Table 4.1. Name and nucleotide sequence of the shRNA probes used in studies involving RNA interference.

4.2.1.1 shRNA transfection of HEK293 cells

Cells were prepared 24 hours prior to shRNA transfection. For Ca2+ imaging experiments and verification by Western blot, transfection was carried out in FluoroDishes and 60 mm sterile petri dishes respectively. These were prepared beforehand as described above. For verification by fluorescent labelling, shRNA transfection was carried out using cells seeded onto poly-D-lysine coated coverslips placed within sterile 30 mm tissue culture dishes as described in Chapter 2.

HA-hTPC2 cells were transfected with the shRNA plasmids using the transfection agent GeneJammer (Stratagene, Ailent Technologies, CA, USA). The quantities of reagents used varied according to the number of dishes that were to be transfected. Therefore the following method describes the transfection of 1 dish of HA-hTPC2 cells and was scaled up as appropriate: 94 µl of sterile, serum-free,

antibiotic-free D-MEM (Opti-MEM; at room temperature) was transferred to a polystyrene tube. 6 µl of GeneJammer transfection reagent was pipetted directly into

the serum-free media and the solution was gently mixed with the pipette tip before being incubated for 5 minutes at room temperature (22°C). 2 µl of the shRNA

plasmid solution (1 µg/µl) was then added to the solution and mixed gently with the

pipette tip. This solution was then incubated for 30 minutes at room temperature. GeneJammer is a polyamine based transfection reagent that is positively charged. Due to their polarity, polyamines are able to bind effectively to nucleic acids, which are negatively charged, in solution. Upon binding to the shRNA plasmid

in solution, particles are formed in which the plasmid is surrounded by the positively charged polyamine molecules. This then allows the plasmid/polyamine particle to interact with the negatively charged plasma membrane of the cell. Following this interaction it is thought that the plasmid / polyamine particles are internalized by the cell via endocytosis (Kanatani et al., 2006; Orth et al., 2008).

A previously prepared dish containing HA-hTPC2 cells was taken and the growth media was removed and replaced with 900 µl of fresh growth media.

Following the incubation of the GeneJammer transfection reagent with the shRNA plasmid, 100 µl of this transfection mixture was added drop-wise and the dish was

gently mixed. The dish was incubated for 8 hours at 37°C, 5% CO2. A further 1 ml

of fresh growth media was then added and the dish was returned to the incubator whereby it was incubated for a further 3-4 days.

4.2.2 Determination of shRNA-mediated knockdown of two-pore channel subtype 2