DNA manipulation

a)Production of plasmid

JM109 competent cells were thawed on ice. 50 µL of cells were transformed with 10 µL

of ligation product or 0.5 µl of plasmid DNA and incubated on ice for 20 to 30 min. Cells were then spread on LB agar plates containing Ampicillin (100 µg/ml). Plates were incubated overnight at 37°C.

(iii)Growth of single colony in suspension

Single colonies were picked using a 200 ml pipette tip. The pipette tip was then partially

submerged in 8 ml of LB medium containing Ampicillin at 100 µg/ml. the inoculated cultures

tubes were incubated overnight at 37°C in a shaking incubator (200 rpm).

(iv)Purification of plasmid DNA

Small quantities: 4 ml of cell suspension were pelleted in 2 ml Eppendorf tubes by

centrifugation at 8,000 rpm for 3 min. The cell pellet was then processed using the QIAprep®

Spin Miniprep kit (Qiagen) according to the manufacturer’s instructions.

Large quantities: 100 to 150 ml of cell suspension were pelleted in 50 ml Falcon tubes by centrifugation for 30 min at 4,000 rpm. The cell pellet was processed using the QIAprep

81 HiSpeed kit (Qiagen) according to the manufacturer’s instruction, ecxept that DNA was eluted in 500 µl of deionised water.

(v)Quantification

The amount of DNA present in each sample was quantified using a 2 µl drop on a spectrophotometer, Nanodrop (Thermo Scientitist).

b)

Amplification of DNA by Polymerase Chain Reaction

Polymerase Chain Reactions (PCR) were performed using the KOD Hot Start polymerase from Merck. Reactions were set up in thin-walled 0.5 ml Eppendorf tubes according to the manufacturer’s instructions. Briefly, the final PCR mix was composed of 1X KOD Hot Start

polymerase buffer, 1.5 mM MgSO4, 0.2 mM of each dNTP, 0.3 µM forward primer, 0.3 µM

reverse primer and 0.02 U/ml KOD Hot Start polymerase. DNA template was added and the final volume was adjusted to 50µl with water.

According to the manufacturer’s protocol, KOD Hot Start polymerase was added only after the initial denaturing step. Routine PCR cycles were performed as follows in a thermal cycler with heated lid:

Initial denaturation 95°C 3 min

Strand separation 95°C 30 sec

x cycles

Annealing According to primer’s Tm 30 sec

Extension 70°C 10 - 25 s/kb

Final extension 70°C 10 min

Hold 10°C ∞

If the PCR was performed using plasmid DNA, PCR products were digested with 10 U of restriction enzyme DpnI (Fermentas) in 1X Tango buffer (Fermentas) for 1h at 37°C.

(i)Standard PCR

Standard PCR was used to amplify specific DNA fragments. Primers were designed to flank each side of the fragment to be amplified in inward orientation such that the 5’-end of the forward primer annealed to the 5’ boundary of the fragment to be amplified and the 5’-end of the reverse primer annealed to the 3’ boundary of the fragment to be amplified. Primers were 18 to 25 nucleotides long. 0.5 µl of plasmid DNA or 2 µl of cDNA were used as template. 25 cycles were performed.

Chapter 2: Materials and methods Methods

82 The size of the product was then checked on an agarose gel as described below.

(ii)Excision PCR

Specific deletions were introduced in the UTRs by excision PCR. For this type of PCR, we used primers flanking each side of the deletion in outward orientation such that the 5’-end of the forward primer annealed to the 3’ boundary of the sequence to be deleted and the 5’- end of the reverse primer annealed to the 5’ boundary of the sequence to be deleted. Primers were 18 to 25 nucleotides long and 0.5 µl of plasmid pT7riboBUNX(+) was used as a template. 25 cycles were performed.

Following digestion by DpnI, PCR products were purified on an agarose gel and ligated as described below.

(iii)Quick-change PCR

This technique allows the introduction of specific point mutations. Primers were designed to contain the mutation and to anneal to the same sequence on opposite strands of the plasmid. Primers were between 35 and 45 nucleotides in length with a melting temperature ≥ 70°C. The desired mutation(s) was located in the middle of the primer and flanked by 10 to 15 nucleotides of correct sequence. Optimally, primers contain 40% of GC and terminate with at least one G or C. 0.5 µl of template was used and 18 cycles were performed.

After digestion by DpnI (without using the Tango buffer), 10 µl of PCR products were transformed into 50 µl of competent cells without further purification.

c)Agarose gel electrophoresis and gel extraction

DNA fragments were separated through a horizontal gel composed of 1X TAE with 0.7-

1% agarose and 0.04 µg/ml ethidium bromide. The gel was submerged in 1X TAE. Samples

containing loading dye (1 volume in 6 volumes) were loaded in wells and a 100 V current was applied for 60 to 100 min.

DNA fragments were visualised on a UV trans-illuminator and excised from the gel, if

needed. DNA was extracted from the gel matrix using the Wizard® SV Gel and PCR Clean-up

System (Promega) according to the manufacturer’s protocol.

d)

Ligation

For ligation of excision PCR products, phosphate groups were added at the 5’-extremity

83 T4 DNA ligase buffer (Invitrogen). The mix was incubated 30 min at 37°C. Then, 1-3 U T4 DNA ligase (Promega) was added.

Ligations of vector and insert were set up in a final volume of 20 or 30 µL. The reactions

contained 50 ng of vector, 50 or 150 ng of insert, 4 or 6 µL of 5X T4 DNA ligase buffer (Promega) and 1 or 3 U T4 DNA ligase (Promega).

Ligations were incubated overnight at room temperature.

When mentioned, the Quick ligation kit from NEB was used according to manufacturer’s instructions.

e)Enzymatic digestion

Endonuclease restriction digests were set up in a final volume of 15 to 30 µl. The reaction contained 1X of the appropriate buffer, acetylated BSA (10 µ g/µl) when needed, 1 or 2 U of enzyme per 1 µg of DNA and was incubated at 37°C for up to 4h.

f)

Transfection of mammalian cells

BSR T7/5 cells were used in transfection experiments. 60 mm-diameter Petri dishes were

seeded 24h prior to transfection with a total of 4x105 cells per dish resulting in ~60% to 70%

confluency.

1 µg of each plasmid DNA to be transfected was added to 350 µL of Opti-MEM in an

Eppendorf tube. For a 60-mm dish, 9 µL of Lipofectamine 2000 reagent (Invitrogen) was

added to 350 µL of Opti-MEM in a haemolyse tube and incubated at room temperature for 5

min. Then, the content of the Eppendorf tube was transferred into the haemolyse tube. The DNA/lipofectamine mix was incubated at room temperature for 20 to 30 min.

The BSR T7/5 monolayer was washed once with 2 ml Opti-MEM before addition of the DNA/Lipofectamine mix. Dishes were then incubated at 37°C for 1h and rocked every 15 min. 4 ml of GMEM – 10% FCS – 10% TPB medium were added and dishes incubated at 33°C.

g)Mini-replicon assay

This assay is based on the one developed by Weber et al. (Weber et al., 2001). BSR T7/5 cells were co-transfected with expression plasmids for the N and L proteins of BUNV, pTM1- BUNN and pTM1-BUNL, and with plasmids encoding a luciferase based mini-genome, for example pT7riboBUNLRen(-). The plasmid pTM1-FF-Luc, expressing the firefly luciferase gene under the control of the T7 promoter was transfected to serve as an internal control.

Briefly, after transfection, pT7riboBUNXRen(-) is transcribed by the T7 polymerase producing viral-like RNA genome sgment. The N protein encapsidates the RNA genome to

Chapter 2: Materials and methods Methods

84 form RNA-N complexes that are recognised by the L protein. The polymerase will synthesise antigenomic and mRNA from the genome template. Renilla luciferase mRNAs are produced only if the viral polymerase can perform transcription and replication.

BSR T7/5 cells were seeded in either a 12-well plate (8.104 cells/well) or in a 24-well

plate (4.104 cells/well). Transfections were done as described above, except that 0.3 µg/well

of each plasmid were used to transfect a 12-well plate or 0.15 µg/well of each plasmid were used to transfect a 24-well plate. 24h post-transfection, cells were lysed and luciferase assay performed using the Dual Luciferase Reporter Assay system (Promega).

Experiments were repeated three times with duplicate or triplicate wells. In each experiment, the values of the firefly luciferase were normalised to the value obtained in mock transfected cells, and the Renilla luciferase values were normalised to the normalised firefly luciferase. Then, the Renilla value of the positive control (or wt) was considered 100% of the relative light activity and all other values were normalised against it to give a percentage. The percentage values of the three repeats were used to calculate the mean and the standard deviation of the mean.