We apply this technique to introduce the cell certain transcription element HNF4a into a human embryonic kidney cell line and investigate its effect on cell proliferation. Success Style and design on the integration techniques To permit web site precise integration of two distinct trans genes we made use of two independent systems. The initial is according to FLP recombinase mediated recombination of FRT websites. By using hygromycin resistance, recombined cell lines could be chosen. Also, loss of lacZ reporter exercise monitors precise recombination with all the FRT integration vector. Conditional transcriptional management from the transgene is achieved by applying the tetracycline inducible process. This process is very well established and com mercially obtainable. As a sec ond and independent technique we formulated FC31 integrase mediated recombination of attP and attB internet sites.
To permit site directed integration of any gene of interest applying the FC31 integrase, we made the docking construct pDOCKING Neo containing the attP recognition web site. This web site is linked in frame to an ECFP neomycin resistance fusion protein as choice marker. Because the attP website is placed selleckchem downstream from the get started codon for ECFP Neo, this marker will likely be inactivated upon certain recombination using the corresponding attB sequence. The incoming attB integration vector is made up of the attB web site fused to a promoter and ATG significantly less puromy cin resistance gene, which can be so activated on distinct recombination using the attP sequence with the docking internet site. The GOI is placed downstream with the puromycin resis tance sequence.
In all experiments we made use of the CMV professional moter and fused the GOI for the L106P mutant from the human FKBP12 protein to reg ulate the expression on the degree of protein stability by Shld1. Generation of double docking HEK293 cell lines The commercially out there Flp In T Rex HEK293 selleck cells include a stably integrated single copy FRT docking internet site for FLP mediated integration. Using this cell line we introduced additionally for FC31 mediated transgene integration the newly generated attP docking web-site that encodes an ECFP Neo fusion gene. Soon after variety with G418 we chosen 24 single clones with blue fluores cence. To find out the quantity of integrated transgene copies we performed true time PCR on genomic DNA and selected the three double docking cell lines 12, 16, and 19 with apparent single copy integration on the docking site for further experiments. Independent integration of inducible fluorescent proteins into various double docking HEK293 cell lines To test regardless of whether independent integration likewise as inde pendent conditional activation of two various GOI might be attained within the double docking cell lines we generated two different integration vectors.