University of Zagreb. Faculty of Science. Department of Biology.
Abstract
ciljem unapređenja sortiranja genetički promijenjenih stanica nedavno je razvijena varijanta sortiranja pomoću magneta. Ona zahtjeva da vektor za genetičku manipulaciju stanica kodira za rekombinantni protein SBP-ΔLNGFR (od engl. Streptavidin Binding Peptide - Low Affinity Nerve Growth Factor Receptor) koji nakon ekspresije lokalizira na membranama stanica. Njegov streptavidin vezujući dio (SBP) tada se usmjerava u izvanstaničan prostor te snažno veže streptavidin na magnetnim kuglicama kojima se vrši separacija stanica. Cilj ovog rada bio je sortirati transfeciranu populaciju stanica FreeStyle 293-F opisanom metodom. Konstruirana su dva donorska plazmida, jedan s modulom SBP-ΔLNGFR, a drugi s mRuby3. Metodom Golden Gate kloniranja, oni su zajedno s modulima dCas9-DNMT3A i gRNA sastavljeni u plazmide kojima su transfecirane stanice. Uspješna transfekcija zabilježena je u 14.5% stanica detekcijom mRuby3 proteina. Metodom imunofluorescencije 24 sata nakon transfekcije potvrđena je lokalizacija SBP-ΔLNGFR na membranama stanica, dok nakon 48 i 72 sata njegova prisutnost više nije zabilježena. Stanice su sortirane prema protokolu Miltenyi Biotec: 24, 48 i 72 sata nakon transfekcije, no pozitivan ishod je izostao. Postupak je ponovljen variranjem nekoliko novih uvjeta separacije no također bezuspješno. S obzirom na negativan ishod sortiranja u raspravi rada predlažemo isprobati nove uvjete istog protokola kao i alternativan protokol s magnetnim kuglicama drugog proizvođača.With the aim of improving genetically modified cell sorting, a variant of magnetic cell sorting has recently been developed. It requires that the vector for genetical manipulation of cells encode for the recombinant protein SBP-ΔLNGFR which is localized on the cell membrane. The protein's streptavidin binding site (SBP) is then directed to the extracellular space where it strongly binds streptavidin on the magnetic particles for cellular separation. The purpose of this paper was to sort transfected FreeStyle 293-F cells by the described method. Two donor plasmids were constructed, one with module SBP-ΔLNGFR and the other with mRuby3. By the Golden Gate cloning, they were assembled with dCas9-DNMT3A and gRNA into plasmids for transfection. Successful transfection was observed in 14.5% cells by detection of the mRuby3 protein. Twenty-four hours after transfection, the SBP-ΔLNGFR localization on cell membranes was confirmed by immunofluorescence, while after 48 and 72 hours its presence was not recorded anymore. Cells were sorted by Miltenyi Biotec protocol: 24, 48 and 72 hours after transfection but without positive outcome. The procedure was repeated by varying several other conditions but also without improvement. We propose to try out new conditions for cell separation of the same protocol as well as an alternative protocol with magnetic particles of another manufacturer
