No public access
master's thesis
CONSTRUCTION OF A PLASMID VECTOR FOR INSERTION OF TARGET SEQUENCES INTO HSV-1 GENOME

Lara Djaković (2016)
University of Rijeka
Department of Biotechnology
Metadata
TitleGENERIRANJE VEKTORA ZA UGRADNJU REPORTER GENA U GENOM HERPES SIMPLEKS VIRUSA 1
AuthorLara Djaković
Mentor(s)Igor Jurak (thesis advisor)
Abstract
Razvojem BAC mutageneze otvoren je novi put manipulacije velikih genoma virusa, uključujući Herpesviridae. Stabilnost, lakoća manipulacije i veliki kapacitet BAC vektora unutar Escherichie Coli označili su prekretnicu omogućivši različite modifikacije, od točkastih mutacija, do insercije i delecije virusnih ili stranih gena. Kako bi imali bolji uvid u replikaciju HSV-1 i njegovo širenje tijekom infekcije, in silico i metodama molekularnog kloniranja, konstruirali smo rekombinantni virus u kojem je EGFP (engl. enhanced green fluorescent protein) ekspresijska kazeta ugrađena u intergensku regiju između sekvenci UL3 i UL4. „En passant“ mutagenezom, jednostavnom i efikasnom lambda Red rekombinacijskom tehnikom, generirali smo HSV-1 LD-pINS i HSV-1 LD-EGFP rekombinantne viruse u dva ključna koraka. U prvom koraku, linearizirane rekombinacijske kazete pInserter-MCS i pInserter-EGFP integrirane su homolognom rekombinacijom u ciljano mjesto HSV-1 pozitivnom selekcijom na kanamicin (BAC LD-pIns-KAN, BAC LD-EGFP-KAN). U drugom koraku, I-SceI endonukleaza pocijepala je mjesto prepoznavanja stvarajući dvostruke lomove DNA (engl. double strand brakes, DSB). Susjedne duplicirane sekvence, prisutne na krajevima dsDNA, poslužiile su kao novi substrat za drugu homolognu rekombinaciju, koja je rezultirala gubitkom kanamicina (BAC LD-pIns, BAC LD-EGFP). Uspješnost ugradnje EGFP ekspresijske kazete u genom HSV-1 potvrdili smo metodom fluorescentne mikroskopije i western blotom. HSV-1 LD-EGFP inficirane stanice i bez prisustva promotora emitiraju dovoljnu razinu fluorescencije potrebne za praćenje virusne replikacije in vitro. Odmicanjem od središta virusnog plaka, gdje je EGFP fluorescencija inficiranih stanica najjače izražena, fluorescencija značajno opada. Ovim radom uspostavili smo jednostavan i izravan sustav kontinuiranog praćenja HSV-1 infekcije in vitro te otvorili mogućnost njegove primjene i na druge HSV-1 sojeve. Daljni napori našeg laboratorija biti će usmjereni ka adaptaciji vektora pInserter-MCS za primjenu npr. miRNA „sponge“ tehnologije kojom bi se razjasnila uloga miRNA u virulenciji.
KeywordsHSV-1 (herpes simplex virus 1) lambda Red recombination pInserter-MCS
Parallel title (English)CONSTRUCTION OF A PLASMID VECTOR FOR INSERTION OF TARGET SEQUENCES INTO HSV-1 GENOME
Committee MembersIgor Jurak (committee chairperson)
Miranda Mladinić Pejatović (committee member)
Antonija Jurak Begonja (committee member)
GranterUniversity of Rijeka
Lower level organizational unitsDepartment of Biotechnology
PlaceRijeka
StateCroatia
Scientific field, discipline, subdisciplineBIOTECHNICAL SCIENCES
Biotechnology
Study programme typeuniversity
Study levelgraduate
Study programmeBiotechnology in medicine
Academic title abbreviationmag. biotech. in med.
Genremaster's thesis
Language Croatian
Defense date2016-09-26
Parallel abstract (English)
Developement of BAC mutagenesis has opened a new avenue for the manipulation of large virus genomes, including Herpesviridae. Stability, relatively simple manipulation and large insert size of BAC constructs within the Escherichia Coli has marked a turning point enabling various modifications like point mutations, insertions and deletions of viral or foreign genes. To better understand the replication of HSV-1 and its spread during infection, in silico and with molecular cloning methods, we constructed a recombinant virus in which EGFP (enhanced green fluorescent protein) expression cassette was inserted into the intergenic region between sequences UL3 and UL4. With „En passant“ mutagenesis, a simple and efficient lambda Red recombination technique, we have generated HSV-1 LD-pINS i HSV-1 LD-EGFP recombinant viruses in two key steps. In the first step, linearized recombination cassettes pInserter-MCS and pInserter-EGFP were inserted via Red recombination into target site using kanamycin positive selection (BAC LD-pIns-KAN, BAC LD-EGFP-KAN). In the second step, I-SceI endonuclease cleaved at its recognition site and created a double strand brakes, DSB. The adjoining duplicated sequences, present at the ends of dsDNA, served as the new substrate for second Red recombination, resulting in loss of kanamycin (BAC LD-pIns, BAC LD-EGFP). Successful integration of EGFP expression cassette into HSV-1 genome was confirmed by fluorescent microscopy and western blot. HSV-1 LD-EGFP infected cells even without promoter presence emit sufficient level of fluorescence, necessary for monitoring of viral replication in vitro. Moving away from the center of viral plaque where EGFP fluorescence of infected cells is expressed the most, fluorescence drops significantly. With this study we established a simple and direct system of continuous monitoring of HSV-1 infection in vitro, and opened the possibility of its application to other HSV-1 strains. Far efforts of our laboratory will be directed to adaptation of vector pInserter-MCS for the application of eg. miRNA sponge technology that would clarify the role of miRNA in virulence.
Parallel keywords (Croatian)HSV-1 (herpes simpleks virus 1) lambda Red rekombinacija pInserter-MCS
Resource typetext
Access conditionNo public access
URN:NBNhttps://urn.nsk.hr/urn:nbn:hr:193:561515
CommitterLea Lazzarich