| Abstract | Cilj istraživanja je razvijanje staničnih linija fibroblasta PS120, bez i s proteinom NHERF2, transfeciranih s mutiranim izmjenjivačima Na+ i H+ izooblika 3 (NHE3). Stanice de se u kasnijim funkcionalnim studijama koristiti za istraživanje mehanizama regulacije NHE3 pomodu Ca2+/kalmodulin ovisne kinaze II (CaMK-II). Metodom usmjerene mutageneze mutirana su na sekvenci HANHE3 potencijalna fosforilacijska mjesta za CaMK-II. Sedam mutacija je provedeno na nativnoj sekvenci HANHE3 (pcDNA/TM7-HANHE3), dok se šest mutacija nalazi na krnjoj sekvenci HANHE3 (pcDNA/TM6-d24HANHE3). Krnja sekvenca HANHE3 (d24HANHE3) je mutanta iz koje je uklonjeno vezno mjesto za CaMK-II, segment dug 24 aminokiseline (Leu586-Arg609). Mutirana su slijededa fosforilacijska mjesta: Ser554, Ser562, Ser607, Ser663, Ser693, Ser694 i Ser810. U ekspresijskom vektoru pcDNA/TM7-HANHE3 mutacije su provođene uzastopno, jedna nakon druge, pri čemu su korištene slijedede metode: reakcija PCR, transformacija bakterija mutiranom HANHE3, umnožavanje bakterija u tekudem mediju, izolacija umnoženog plazmida iz bakterija i sekvenciranje. Ekspresijski vektor pcDNA/TM6-d24HANHE3 je konstruiran u dva koraka. U prvom je pomodu krnje sekvence d24HANHE3 konstruirana mutanta pcDNA/TM2-d24HANHE3 unutar koje su mutirani Ser554/Ser562 u Ala554/Ala562. U drugom koraku su najprije restrikcijskom endonukleazom PmaCI pocijepani pcDNA/TM7-HANHE3 i pcDNA/TM2-d24HANHE3 u po dva DNA isječka, duži vektorski dio i kradi umetak. Vektorski dio (f-pcDNA/TM4-HANHE3) izoliran iz pcDNA/TM7-HANHE3 sadržavao je četiri mutacije, dok umetak (fTM2-d24HANHE3) izoliran iz pcDNA/TM2-d24HANHE3 sadržavao dvije mutacije i deleciju veznog mjesta. Zatim je povezivanjem vektora i umetka pomodu ligacije konstruiran ekspresijski vektor pcDNA/TM6-d24HANHE3. Ekspresijski vektori su postupkom transfekcije unijeti u stanične linije fibroblasta PS120 bez i s proteinom NHERF2. Selekcijskim antibiotikom G418 selektirane su uspješno transfecirane stanice, a zakiseljavanjem transfeciranih staničnih linija selektirane su stanice s funkcionalnim mutiranim proteinom NHE3 na površini stanica. |
| Abstract (english) | Goal of this research was development of PS120 fibroblast cell lines transfected with mutated Na+/H+ exchanger isoform 3 (NHE3), with or without NHERF2. Developed cell lines will be used in future studies to examine Ca2+/calmodulin dependent kinase II (CaMK-II) regulation mechanisms of NHE3. Site directed mutagenesis was used on a gene sequence for HANHE3 to develop point mutations on potential phosphorylation sites for CaMK-II. Seven mutations were made on a native HANHE3 sequence (pcDNA/TM7-HANHE3), while six mutations were made on HANHE3 sequence that has 24 amino acids long (Leu586-Arg609) deletion of CaMK-II binding site (d24HANHE3). Mutations were made on following amino acids: Ser554, Ser562, Ser607, Ser663, Ser693, Ser694 and Ser810. Mutations on pcDNA/TM7-HANHE3 where made successively, one after the other, using: PCR reactions, bacterial transformation with mutated sequence, amplifying of bacteria in liquid medium, isolation of amplified product, and sequencing. Construction of pcDNA/TM6-d24HANHE3 was a two-step process. First, mutations Ser554Ala and Ser562Ala were made on d24HANHE3 as a template (pcDNA/TM2-d24HANHE3). Second, both pcDNA/TM7-HANHE3 and pcDNA/TM2-d24HANHE3 were cut using PmaCI restriction enzyme to acquire insert and vector needed for construction of pcDNA/TM6-d24HANHE3. Vector (f-pcDNA/TM4-HANHE3) was isolated from pcDNA/TM7-HANHE3, and it had four mutations, while insert (fTM2-d24HANHE3) was isolated from pcDNA/TM2-d24HANHE3 and had two mutations of CaMK-II potential phosphorylation sites and the deletion of CaMK-II binding site. Then using ligation, vector and insert were connected to form pcDNA/TM6-HANHE3. Mutated plasmids pcDNA/TM7-HANHE3 and pcDNA/TM6-HANHE3 were transfected into PS120 fibroblast cell lines with or without NHERF2. Cells were selected with antibiotic G418 to make sure that transfection was successful. Acid loading of transfected cells was performed to select cells with expression of functional mutated NHE3. |
