RNA interference (RNA i )

RNA interference (RNA_i) is a sequence-specific posttranscriptional, gene silencing method to analyse gene function in various organisms. The effectors of RNAi are small interfering RNAs (siRNAs) that are processed from longer precursors (e.g. short hairpin shRNA).

Short hairpin RNA molecules contain a sense and antisense sequence from a target gene that are connected by a hairpin loop. This structure is cleaved by a ribonuclease known as dicer. The resulting siRNA is incorporated into the RNA induced silencing complex

(RISC) where sense and antisense strands are separated. The RISC containing the

antisense strand seeks and binds to complementary mRNA sequences. The binding of the complex to the target mRNA induces its degradation.

6.3.1 Short hairpin RNA (shRNA) cloning

RNA_i experiments were performed using the small hairpin shRNA expression vector pDLDU6 [325]. This plasmid (4264bp) is based on the pTRE shuttle (BD Clontech, Basel, Switzerland) but the promoter region (CMV) has been replaced by the RNA polymerase III U6 promoter to ensure permanent expression of the shRNA. It contained a kanamycin resistance cassette as well as a Pme I and Xba I restriction site allowing the insertion of short hairpin shRNA sequences. Three 21-nucleotide cytoplasmic aspartate aminotransferase shRNA (shRNAcAAT) target sequences were identified. The targeted sense and antisense sequences are listed in the table below:

shRNA Sequence CDS

cAAT 1 sense 5’-GCC GGT TCT GGT CTT TAA GCT-3’ 84-104 antisense 5’-AGC TTA AAG ACC AGA ACC GGC-3’

cAAT 2 sense 5’-GCC GAC CTG GGA GAA CCA TAA-3’ 456-476 antisense 5’-TTA TGG TTC TCC CAG GTC GGC-3’

cAAT 3 sense 5’-GGC TGA CCG GAT TCT GAC CAT-3’ 1023-1043 antisense 5’-ATG GTC AGAATC CGG TCA GCC-3’

Table 6.1: shRNA sense and antisense sequences targeting three different regions in the coding sequence (CDS) of the cytoplasmic aspartate aminotransferase (cAAT) gene.

For each shRNA a forward and a reverse complement was generated. Each complement was composed of a 21 nucleotide sense and antisense sequence connected by a 6 nucleotide short hairpin loop. The loop contained either a Xho I (cAAT 1 & 3) or a Pst I (cAAT 2) restriction site. This site was used to confirm successful cloning of the short shRNA construct into the vector). The 3’-end of the forward complement ended with a string of T’s while the reverse complement contained an additional GATC overhang creating a Xba I restriction site. The forward and reverse complement were annealed and ligated to the Pme I and Xba I sites of the pDLDU6 plasmid followed by transformation, plasmid isolation and verification of successful insertion:

Different steps of this procedure

1. Digestion of pDLDU6 with Pme I and Xba I

2. Oligonucleotide annealing

The forward and reverse complement were diluted to a stock concentration of 40pmol/ul. 10µl of each oligonucleotide was mixed with 20µl of deionised water.

The preparation was heated for 5min at 95°C in a heating block. The oligonucleotide mixtures were then placed at room temperature for 1.5h. Slow reduction of the temperature allows for the annealing of matching oligonucleotide pairs.

3. Ligation The annealed sense and antisense oligonucleotides are ligated into the digested pDLDU6 plasmid

4. Transformation

The ligation reaction was transformed into DH5α competent E.coli and kanamycin-resistant transformants were obtained.

5. Verification of successful insertion of shRNA oligonucleotides

To identify recombinant plasmids, first restriction analysis was performed using either the restriction enzyme Xho I or Pst I that were introduced in the shRNA construct (see above chapter 6.3.1). Recombinant plasmids showed two DNA fragments of either 1080 and 3180bp (Xho I) or 820 and 3460bp (Pst I).

Orientation and junctions of the insert were additionally verified by sequencing (Service of the Faculty of Medicine, University of Geneva).

6.3.2 INS-1E cell transient transfection

Constructs (pDLDU6-shRNA) were transiently co-transfected into INS-1E cells along with a reporter GFP-expressing vector (Clontech) in order to demonstrate sequence-specific posttranscriptional, gene silencing. This approach allows the selection of GFP-positive shRNA expressing cells. To do so, Lipofectamine^TM 2000 reagent (Invitrogen Ref 11668) was used. One day before transfection, INS-1E cells were seeded on polyornithine-coated-6-well plates (1x10⁶ cells per well) and cultured overnight in RPMI-1640

containing 11mM glucose (~70% confluence). Transfections were performed in duplicate.

6µg of recombinant pDLDU6 plasmid and 3µg of GFP-expressing vector (ratio shRNA plasmid to reporter GFP vector 1:2) were diluted in 500ul OPTI-MEM® (Invitrogen Ref 31985-062) and incubated at room temperature for 5- 10min. For each duplicate, 20µl of lipofectamine (2µg/ml) was diluted in 500ul OPTI-MEM® and also incubated at room temperature for 5-10min. DNA samples and lipofectamine were mixed and left at room temperature for 30min. INS-1E cells were washed twice with pre-warmed PBS devoid of Ca²⁺ and Mg²⁺ followed by the addition of 500ul of OPTI-MEM® and 500µl of the lipofectamine-plasmid complex. 4h post-transfection 1ml of RPMI-1640 medium was added. The next day medium was replaced by fresh RPMI-1640 medium and cells were cultured at 37°C for another 48h. 72h post-transfection, fluorescence-activated cell sorting (FACS analysis) was performed in order to separate transfected (GFP-/shRNA expressing cells) from untransfected cells. For this purpose, INS-1E cells were trypsinized as described above. The cell suspension was transferred into an eppendorf tube, centrifuged (900rpm, 3min) and the cell pellet was resuspended in 0.5ml KRBH buffer containing 0.25% BSA and 2.5mM glucose. Duplicates were pooled and filtered through a 70µM nylon cell strainer (BD Falcon Ref 352350). GFP/shRNA-expressing cells were sorted on a FACSVantage SE (Becton Dickinson). Untransfected cells were also collected (control cells). Finally sorted cells were centrifuged at 1600rpm for 5min and the cell pellet was either used immediately for total RNA isolation or resuspended in 500µl RNA later stabilization reagent (Qiagen Ref 76104) for storage of the samples at -70°.

6.3.3 RNA isolation & reverse transcription

Total RNA was extracted from sorted transfected and untransfected INS-1E cells using the Qiagen RNeasy mini kit (Qiagen Ref 74104) and RNase-free DNase set (Qiagen Ref 79254). In parallel, total RNA was also isolated from 2x10⁶ INS-1E cells which were used as a standard during quantitative real-time PCR reactions. DNA shredder columns (Qiagen Ref 79654) were utilized for cell lysis. 2 µg of total RNA were used for cDNA synthesis by SuperScript II Reverse Transcriptase (Invitrogen Ref 18064-022). Random hexamers were purchased from Invitrogen (Ref 48190-011). They were used at a concentration of 150ng/ul and mixed on ice.

The reaction was incubated at 65°C for 5min to allow random hexamers to anneal to the mRNA. Samples were then put on ice to maintain the annealed state. Reverse

transcriptase SuperScript II, dNTP’s, DTT (dithiothreitol) and buffer was added to obtain cDNA.

6.3.4 Quantitative real-time PCR (QT-PCR)

The effect of the shRNA’s on _cAAT mRNA expression was monitored by quantitative real-time PCR (QT-PCR). Primers for cAAT and cyclophilin (housekeeping gene) were designed using the Primer Express Software (Applera Europe, Rotkreuz, Switzerland) (Table 6.2).

Primer Name Gene Sequence

rCAAT-5' cAAT GCT GAC CGG ATT CTG ACC AT

rCAAT-3' cAAT CCC GGG AGT CTT GAG AGC TT

Cyclophilin-5’ Cyclophilin TCA CCA TCT CCG ACT GTG GA Cyclophilin-3’ Cyclophilin AAA TGC CCG CAA GTC AAA GA

Table 6.2: Sequences of primers used for quantitative real-time PCR (QT-PCR) analysis.

QT-PCR was performed using an ABI 7500 Sequence Detection System (Applied Biosystems AB) and PCR products were quantified using the SYBR Green Core Reagent kit (AB Applied Biosystems Ref 4367659) [326]. The SYBR Green PCR Master Mix (Ref 4367659) combines SYBR Green I dye, AmpliTaq Gold® DNA polymerase, dNTPs and optimized buffer. SYBR I Green fluorescence appears as the dye binds to double-stranded DNA. Fluorescence signal intensity therefore increases during the formation of the specific PCR product. Initial generation of the PCR product to an amount detectable by SYBR Green (threshold) depends on the initial concentration of target cDNA.

Amplifications were performed in duplicate for each transcript and mean values were normalized to the mean value of the reference mRNA cyclophilin.

For QT-PCR reactions, a primer mix for each gene (cAAT/cyclophilin) was prepared containing 5’-rCCAT/cyclophilin and 3’-rCCAT/cyclophilin. The final concentration of the primers corresponded to 300nM. The primer mix was added to cDNA and PCR reactions were performed in the presence of SYBR Green PCR master mix.

PCR amplification was performed for 50 cycles using the ABI 7500 Sequence Detection System

The cycle threshold (CT) for _cAAT and cyclophilin was determined in every sample. CT defines the numbers of cycles required for the fluorescent signal to cross the threshold.

CT values are proportional to the amount of the target DNA in the sample. In order to determine _cAAT and cyclophilin cDNA amounts in every sample a standard curve for both genes generated by plotting the CT values of each standard INS-1E cDNA against the corresponding cDNA amount [log(ng cDNA)]. cAAT mRNA expression was normalized to

the housekeeping gene cyclophilin. Expression data were compared between shRNA transfected GFP-positive (shRNAcAAT) and GFP-negative control cells after FACS sorting.

6.3.5 Construction of recombinant adenoviral DNA

6.3.5.1 PI-Sce I/I-Ceu I digestion of recombinant pDLDU6/shRNA plasmid

The U6/shRNA cassette of the recombinant pDLDU6 was transferred into the promoter-deficient Adeno-X^TM viral DNA (Clontech) to generate recombinant adenovirus (AdshRNAcAAT). To excise the U6/shRNA cassette from pDLDU6-shRNA vector, the restriction enzymes PI-Sce I and I-Ceu I (Clontech) were used. The preparation was incubated at 37°C for 3 to 5h. The gel loading buffer contained 0.1% SDS to strip the restriction enzymes, which otherwise remain bound to the DNA and prevent proper separation in the TBE agarose gel. Two bands corresponding to U6/shRNA (~1400bp) and backbone vector (~2800bp) were observed (Figure 6.1). The U6/shRNA cassette (~1400bp) was extracted using the ‘MinElute Gel Extraction Kit’ (Qiagen Ref 28604).

Figure 6.1: Excision of U6/shRNA expression cassette from recombinant pDLDU6-shRNA plasmid. The restriction enzymes PI-Sce I and I-Ceu I were used to excise the U6/shRNA expression cassette (~1400bp) from recombinant pDLDU6 plasmid.

6.3.5.2 Subcloning of U6/shRNA expression cassette into Adeno-X^TM genome

The U6/shRNA cassette was ligated to Adeno-X^TM viral DNA (~31kb) (Clontech Ref 631026) using T4 DNA ligase. The ligation product is a circular recombinant E1/E3-deleted adenoviral genome that contains a ColE1 origin of replication and an ampicillin resistance marker for propagation and selection in E.coli. The DNA was precipitated using ammonium acetate and EtOH as well as glycogen as a carrier (1µl glycogen, 0.5 Volume 7.5M ammonium acetate, 2.5 Volume ice-cold 100% EtOH).

6.3.5.3 Transformation of putative recombinant Adeno-X^TM DNA in electrocompetent DH10B cells

The Adeno-X^TM viral DNA ligation product was electroporated into DH10B bacteria in a MicroPulser Electroporator (Bio-Rad laboratories Ref 165-2100). Ampicillin-resistant transformants were selected on LB-agar containing 100µg/ml ampicillin.

6.3.5.4 Analysis of putative recombinant Adeno-X^TM plasmid DNA

The primers p206 and p207 (Table 6.3) were used for the PCR screening of U6/shRNA cassette positive ampicillin resistant clones. Ampicillin-resistant clones were transferred into 40µl of H2O. 35 µl of this bacteria suspension were utilized for recombinant Adeno-X^TM DNA amplification while the remaining 5µl were analysed for successful insertion of U6/shRNA expression cassette by PCR reaction. Intact bacteria were heated for 5min at 95°C in the Thermocycler to allow access of the Taq DNA polymerase to the template DNA followed by a standard PCR reaction including heating, primer annealing and elongation of DNA for 35 cycles.

Primer Name Plasmid [base] sequence

Forward primer (p206) 224-242 TGC GCT GCT TCG CGA TGT A Reverse Primer (p207) 677-698 GGT GTG GGA GGT TTT TTA AAG C Table 6.3: Sequence of primers used for PCR screening

A 500bp band was obtained for U6/shRNA positive clones. Only PCR-positive clones were further amplified. Clones carrying the intact AdenoX vector displayed a 32kb DNA on a 1% TBE agarose gel.

6.3.6 Production of recombinant infectious Adenovirus

Recombinant infectious adenovirus was produced by transfecting HEK293 cells with recombinant Adeno-X^TM viral DNA. Before Adeno-X^TM DNA can be packaged, the recombinant plasmid must be linearized by digesting with Pac I. The Adeno-X^TM plasmid contains two Pac I restriction sites, which are located at both ends of the viral genome (to allow the formation of the replication complex). Two DNA fragments of 3kb (Ampicillin cassette) and ~29kb Adeno-X^TM backbone DNA containing the shRNA construct were obtained after Pac I digestion. This linearized DNA constitutes the viral genome that can be packaged to form viral particles in HEK293 cells.

6.3.6.1 Pac I digestion

Linearized plasmid DNA was precipitated as described above (chapter 6.3.5.2). The precipitated DNA was resuspended in H2O to be used for calcium phosphate (CaPO4) transfection of HEK293 cells.

6.3.6.2 CaPO₄ transfection

One day before transfection HEK293 cells were plated at a density of 1-2x10⁶ cells per 60mm petri dish (adherent). For optimal results, the cells should be 50-70% confluent at the time of transfection. The CaPO₄ transfection was performed in duplicate using the calcium phosphate precipitation method according to the manufactory guidelines (Clontech CalPhosTM Mammalian Transfection Kit Ref N° 631312). The final volume of DNA-CaPo₄ mix was increased to 500ul and added to HEK293 cells. Virus infection and formation by HEK cells results in a cytopathic shock. Infected cells typically remained intact but round up and may detach from the petri dish. Usually the CPE is only observed after several rounds of infection as viral titer is very low at the beginning. Even if no CPE was observed cells were lysed after ~5 days. Cells were detached from the culture dish and collected by centrifugation. The cell pellet was resuspended in 300µl PBS and lysed by freeze and thaw cycles. The cell debris was briefly centrifuged and either stored at -80°C or used to infect newly plated HEK293 cells. Usually it takes several rounds of HEK293 infections until the CPE becomes evident (3-4 rounds). Each viral stock preparation should be clearly named (primary amplification, second amplification etc.).

6.3.6.3 Preparation of high-titer recombinant adenoviral stocks

Lysates from four 15cm Petri dishes of HEK293 cells were prepared to obtain enough particles for CsCl virus isolation. The volume of concentrated virus lysate was adjusted to 6ml with 10mM TrisHCl pH8.0.

6.3.7 Adenovirus purification

Recombinant adenovirus was purified using two consecutive caesium chloride gradients of different density (ρ(CsCl)=1.43 & ρ(CsCl)=1.34; Applichem N° A1098).

In the first purification step 5ml of CsCl solution (density 1.43) were filled in an ultraclear tube (Beckman 9/16 x 3.5 UC Ref 344059). The virus solution was carefully layered on top followed by an ultracentrifugation (Sw41Ti) at 4°C and 25’000rpm for 1h.

After this centrifugation step three layers were observed. The upper layer containing accumulated lipids was carefully removed. Two further bands were noted: a pink protein-rich band and a lower grey band containing the virus. Sometimes the protein and virus layer did not form two distinct bands. Therefore, caution was demanded when the virus was collected (1-2ml). The virus fraction was transferred into a 15ml falcon tube and mixed with 8ml of CsCl solution (density 1.34). The virus-CsCl mix was transferred into a new ultraclear Beckman tube. A second centrifugation was performed at 4°C and 30’000rpm for 19-20h. At the end of the centrifugation a nicely visible band was observed. The virus layer was collected (1-2ml) and placed on ice. Finally, the virus was isolated by PD-10 sephadex column fractionation. The PD-10sephadex column (Amersham Bioscience Ref 17-0851-01) was equilibrated with 25ml of PBS w/o Ca²⁺ and Mg²⁺. 24 eppendorf tubes were prepared to collect the different virus fractions. The virus solution was loaded on the equilibrated PD-column and 8 drop fractions were collected in 1.5ml eppendorf tubes. Once the virus solution penetrated into the column PBS w/o Ca²⁺

and Mg²⁺ was added while collecting the fractions. The fractions were placed on ice and 5µl of each fraction was added onto a nitrocellulose sheet to determine relative viral protein amount. Viral protein was visualized by addition of Ponceau solution. Once staining was complete Ponceau solution was washed of with deionized water. Fractions containing the highest amounts of viral protein were pooled. 1/100 volume of 10% BSA and 1/10 volume of glycerol (Fluka Ref 49767) was added to the isolated virus and small aliquots (5-20µl) were frozen in liquid nitrogen and stored at -70°C.

6.3.8 Adenovirus titer determination

The adenoviral titer was determined according to the manufacture’s guidelines of the Adeno-X Rapid Titer Kit (Clontech Ref 631028). The primary anti-Hexon antibody was used at a concentration of 1:2000 (in PBS/BSA) whereas the second rat anti-mouse-HRP antibody was diluted 1:100. The DAB enhanced liquid system (Sigma Ref 3939) was used (1 drop of DAB liquid chromogen solution were added to 1ml of DAB liquid buffer solution) to detect virus-infected HEK293 cells. A minimum of three fields of brown positive cells were counted using a microscope with a 20x objective. The infectious units per ml [ifu/ml] were calculated according to the following formula:

(infected cells/field) x (fields/well) volume virus (ml) x (dilution factor)

The 20x objective corresponds to 594 fields in a 12well plate.

6.3.9 Efficiency of adenovirus

The aim was to identify the optimal virus infection concentration where cytopathic effects (CPE) were minimal but gene repression was maximal. To do so, 5x10⁵ INS-1E cells were seeded on a polyornithine-coated-12-well plate and cultured in 2ml standard RPMI-1640 medium at 37°C overnight. Various infection titres were tested ranging from 0ifu/cell to 160ifu/cell. All concentrations were tested in duplicate. For viral infection, RPMI-1640 medium was removed and 300µl of fresh growth medium was added. Different virus concentrations were added to the cells and incubated at 37°C for 1.5h. At the end of the incubation, the supernatant was removed and replaced by fresh RPMI-1640 medium. The CPE was observed periodically. 48h after infection total RNA was isolated and cDNA was synthesized from infected and non-infected INS-1E cells. The efficiency of cAAT downregulation was tested by QT-PCR.

6.3.10 Adenoviral infection of INS-1E cells

To analyse the specificity of cAAT repression and to exclude physiological artefacts induced by viral infection, a control adenovirus expressing the green fluorescence protein under the control of the rat insulin promoter (AdRIPGFP) was used for metabolite and

hormone assays (kindly provided by Andreas Wiederkehr, University Medical Center, Geneva).

24 h before infection, 5 10⁵ INS-1E cells were seeded on polyornithine-coated-24-well plates (Falcon Ref 353047). The viral infection was performed as described in chapter 6.3.9. 40ifu/cell were used. 18h before the hormone and metabolite assay RMPI-1640 medium supplemented with 11mM glucose was replaced by RMPI-1640 containing only 4mM glucose (to achieve a better GSIS response).