Irina Callens 18/06/2015 EXPRESSION AND ROLE OF A CYPRINID HERPESVIRUS-3 MICRORNA

INDEX  Cyprinid Herpesvirus-3  MicroRNAs  Methods  Results  Conclusion  Future

 Infects carps and koi.  Leads to drastic consequences in the aquaculture.  One of the main cultivated fish for human consumption;  Produced and stocked into fishing areas for angling purpose;  Koi’s are probably the most expensive freshwater fish.  Has a mucosal route of infection.  Infects fish only when the water temperature is between 18 and 28°C. CYPRINID HERPESVIRUS-3

 Vaccination based on the fact that infection only happen when temperature is between 18 and 28°C.  The protection is observed in only 60%;  ‘Vaccinated’ carps are a potential source for unexposed carp;  Increasing the water temperature makes the fish more susceptible to secondary infections.

MICRORNAS  Small non-coding, single-stranded RNA molecules.  Play a regulatory role in numerous and diverse cellular processes such as immune function, apoptosis and tumor genesis.  MR5057-miR-3p is the most highly expressed miRNA discovered in CyHV-3 in vitro.  3’UTR and mRNA contains a binding site for this miRNA.  When it binds it down-regulates the expression of dUTPase  degradation of dUTPase mRNA.

 Back to back mutagenic PCR  DpnI and Ligase treatment  Making calcium competent cells  Transformation  Identification of recombinant clones by PCR and restriction digest  Plasmid purification METHODS

Identification of recombinant clones by PCR  Lane 1: 100 bp ladder  Lane 2: Positive control (non- mutated plasmid)  Lane 3: Colony 1  Lane 4: Colony 2  Lane 5: Colony 3  Lane 6: Colony 4  Lane 7: Colony 5  Lane 8: Non template control RESULTS

Identification of recombinant clones by PCR and restriction digest (1)  Lane 1: 100 bp ladder  Lane 2: Positive control (non- mutated plasmid)  Lane 3: Colony 1  Lane 4: Colony 2  Lane 5: Colony 3  Lane 6: Colony 4  Lane 7: Colony 5  Lane 8: Non template control

Identification of recombinant clones by PCR and restriction digest (2)  Lane 1: 100 bp ladder  Lane 2: Non-mutated plasmid, undigested  Lane 3: Colony 6  Lane 4: Colony 7  Lane 5: Colony 8  Lane 6: Colony 9  Lane 7: Colony 10  Lane 8: Non template control RESULTS

Result of the PCR and restriction digest to confirm the mutated plasmid in the plasmid prep (1)  Lane 1: 100 bp ladder  Lane 2: Non-mutated plasmid, PCR product  Lane 3: Non-mutated plasmid, PCR product + restriction digest  Lane 4: Colony 6, PCR product  Lane 5: Colony 6, PCR product + restriction digest  Lane 6: Colony 7, PCR product  Lane 7: Colony 7, PCR product + restriction digest  Lane 8: Non template control RESULTS

Restriction digest with different enzymes  Lane 1: 1 kb ladder  Lane 2: Wild type plasmid, EcoRI  Lane 3: Colony 6, EcoRI  Lane 4: Wild type plasmid, BamHI  Lane 5: Colony 6, BamHI  Lane 6: Wild type plasmid, HindIII  Lane 7: Colony 6, HindIII RESULTS

PCR with dilution of the possible mutated plasmid  Lane 1: 100 bp ladder  Lane 2: A dilution of the plasmid RESULTS

Result of the PCR and restriction digest to confirm the mutated plasmid in the plasmid prep (2)  Lane 1: 100 bp ladder  Lane 2: Non-mutated plasmid, PCR product  Lane 3: Non-mutated plasmid, PCR product + restriction digest  Lane 4: Colony 6, PCR product  Lane 5: Colony 6, PCR product + restriction digest RESULTS

CONCLUSION  The aim of the project was to mutate the miRNA binding site.  A positive result before the plasmid purification, but negative after it.  To much DNA loaded?  There went something wrong during the plasmid purification?  RsaI enzyme did not work?  Human fault?  Possible solution: insert the mutant dUTPase commercially synthesized into the same recombinant vector used in this study.

FUTURE  Mutant plasmid transfected into HEK293 cells with a M5057-miR-3p mimic and a negative control (the non- mutated plasmid).  Total proteins are prepared from transfected cells and electrophoreses by SDS-PAGE.  In order to see the level of recombinant dUTPases produced from the plasmids, the gel is blotted onto nitrocellulose and probed with an anti-tag antibody. The antibody can bind to the recombinant dUTPase.

FUTURE

Irina Callens 18/06/2015 EXPRESSION AND ROLE OF A CYPRINID HERPESVIRUS-3 MICRORNA