For the expression study, human GFAP was PCR-amplified from GFAP cDNA (NCBI accession number BC013596, Dharmacon, Lafayette, CO) with specific primers (Table 1), and the resulting PCR product was cloned into the BamHI/EcoRV sites of the pCS4 + −3xFLAG-P2A vector [27]. p.Arg79Cys, p.Arg79His, p.Arg239Cys, p.Arg239His and p.Asp128Asn mutations were individually inserted into the WT GFAP construct by site-directed mutagenesis with specific primers (Table 1). For zebrafish study, the zebrafish gfap regulatory elements (7.4 kb) [25] were cloned into the BglII/SalI sites of a mini-Tol2 (T2AL200R150G) plasmid [28]. EGFP and human GFAP C-terminally fused to a FLAG epitope were then sequentially cloned into the resulting construct (Fig. 2b). All plasmids constructed were verified by DNA sequencing (Macrogen, Daejeon, Korea).
Table 1 Sequences of primers (5′ → 3′) used to construct plasmids encoding various human GFAP alleles
Allele Sequences
WT Forward: TAGTAGGATCCATGGAGAGGAGACGCATCAC
Reverse: TAGTCGATATCATCATCACATCCTTGTGCTCCTGCTTG
p.Arg79Cys Forward: GAGATGATGGAGCTCAATGACtGCTTTGCCAGCTACATCGAG
Reverse: CTCGATGTAGCTGGCAAAGCaGTCATTGAGCTCCATCATCTC
p.Arg79His Forward: GAGATGATGGAGCTCAATGACCaCTTTGCCAGCTACATCGAG
Reverse: CTCGATGTAGCTGGCAAAGtGGTCATTGAGCTCCATCATCTC
p.Asp128Asn Forward: GAGAGCTGCGGCTGCGGCTCaATCAACTCACCGCCAACAG
Reverse: CTGTTGGCGGTGAGTTGATtGAGCCGCAGCCGCAGCTCTC
p.Asp157Asn Forward: GCAGAAGCTCCAGaATGAAACCAACCTG
Reverse: CAGGTTGGTTTCATtCTGGAGCTTCTGC
p.Arg239Cys Forward: CAGCCCTGAAAGAGATCtGCACGCAGTATGAGGCAATG
Reverse: CATTGCCTCATACTGCGTGCaGATCTCTTTCAGGGCTG
p.Arg239His Forward: CAGCCCTGAAAGAGATCCaCACGCAGTATGAGGCAATG
Reverse: CATTGCCTCATACTGCGTGtGGATCTCTTTCAGGGCTG
Lower case indicates mutated nucleotides
Fig. 2 Protein expression levels of mutant alleles were comparable to that of WT GFAP. a HEK293T cells were transfected with plasmid encoding EGFP or indicated alleles of GFAP C-terminally fused to a FLAG epitope, and processed for Western blotting with anti-FLAG antibody. Anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibody was used as a loading control. b Quantitation of GFAP band intensity in (a) normalized to GAPDH band intensity (n = 3). NS: not significant