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Team:Potsdam Bioware/Project/Part Virus
From 2012.igem.org
Part Virus
Work in progress
Introduction
In our project AAV-virus acts as a selectionsystem for CHO-cells. The CHO-cells express antibodies presenting them on their surface. To check the Antibodies being right expressed we focused on two different selectionsystems with two different antigens presented from recombinant AAV-Virus. After a stable formation of antigen-antibody-komplex the virus infect the cells with a gene necessery for selection.
AAV-Virus as a selectionsystem
The first Selectionsystem is based on the expression of several fluorescence proteins. The CHO-cells present on their surface GFP-nanobody, which binds both GFP and YFP with a high affinity and high specifity. The recombinant AAV-Virus carries on its surface YFP to target specifically the CHO-cells with GFP-nanobodies and to infect them.
To examine the effect of the enzyme AID stably trasfected in the CHO-cells to induce the hypersomatic mutation, we designed a recombinant virus with CFP on its surface. GFP-nanobody expressed on CHO-cells binds YFP, but not the closely realted CFP. Our goal was to show the AID mutates the GFP-nanobody to an Antibody with a higher affinity to the CFP. Formation of a stable antigen-antibody complex enables the virus binding to the cell and infect it with a mcherry-signal. By excitation with the UV-light the infected cells could be visualised by shining with red colour.
The second selection system is based on the Virus giving the cells a survival signal by infecting them. Virus with the same CFP-antigen on the surface couldn't bind to the GFP-nanobody. Altered by the AID GFP-nanobody binds CFP linked to the virus following by the infection with Kanamycinresistance genecassette. The Kanamycin/Neomycin gene codes for the aminoglycoside phosphotransferase enzyme, which inactivates by phosphorylation the aminoglycoside antibiotics such as geneticin (G418). Cultured in the correct Geneticin concentration, which was determined by titration against nontransfected controls, selection for stable transfectants is possible.
