TITLE: Cloning and mutagenesis
AUTHORS: Federico Herrera

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>We generated a human-GFAP-EGFP fusion construct by randomly inserting the coding region of EFGP into the coding region of hGFAP (A). Briefly, in the presence of a transposase protein, a 1902 bp transposon containing the coding region for EGFP and kanamycin resistance (Kan</span><span style = "vertical-align:super;">r</span><span>) was inserted randomly into a pcDNA3.1/Ampicillin(Amp</span><span style = "vertical-align:super;">r</span><span>) resistant target plasmid containing the coding region for human GFAP. Successfully transposed plasmids carried both Amp</span><span style = "vertical-align:super;">r</span><span> and Kan</span><span style = "vertical-align:super;">r</span><span> as selection markers. The transposon insertion was confirmed by PCR-based colony screening using primers specific for GFAP. Positive colonies were then digested with Srf1 to remove the Kan</span><span style = "vertical-align:super;">r</span><span> region, and the resulting plasmids were transfected into U251 cells and tested for fluorescence by means of flow cytometry and fluorescence microscopy. Once confirmed the efficacy of the EGFP insert, we tried unsuccessfully to exchange it for a Halo tag and bimolecular fluorescence complementation (BiFC) tags Venus 1 (amino acids 1-158), Venus 2 (amino acids 158-238)(B).</span></div><div class = "text-block"><span>For the mGFAP-Halo construct, the host vector was obtained by substitution of the EGFP tag from a pEGFP-N3-mGFAP plasmid for a HaloTag using </span><span style = "font-style:italic;">BamHI </span><span>and </span><span style = "font-style:italic;">NotI </span><span>restriction sites (C). The HaloTag was obtained by PCR-amplification of the pZH504Halo-Cro plasmid using specific primers and carrying </span><span style = "font-style:italic;">BamHI </span><span>and </span><span style = "font-style:italic;">NotI </span><span>restriction sites. PCRs were done using 20 ng of template DNA, 0,2 µM of cloning primers and 200 µM of dNTPs in a total volume of 50 µl containing 1 unit of Phusion DNA polymerase and the corresponding amplification buffer. The thermocycling conditions were the following: denaturation at 98ºC for 30 sec followed by 30 cycles at 98ºC for 10sec; 65ºC for 30s; 72ºC for 30 sec (30s/kb, as specified by the manufacturer) and a final extension at 72ºC for 10 min. </span></div><div class = "text-block"><span>Single p.Arg236His(R236H) and p.Arg239Cys(R239C) mutations were individually inserted by site-directed mutagenesis into the wild-type mGFAP and hGFAP constructs, respectively. PCRs were carried out using 25 ng of template DNA, 0,2 µM of mutagenesis primers and 250 µM of dNTPs in a total volume of 50 µl containing 2,5 units of </span><span style = "font-style:italic;">Pfuturbo </span><span>DNA polymerase and the corresponding amplification buffer. The following thermocycling conditions were used: denaturation at 95ºC followed by 12 cycles at 95ºC for 30 sec; 55ºC for 1 min; 68ºC for 8 min (1 min/kb, as specified by the manufacturer) and a final extension at 68ºC for 5 min.</span></div></div>

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