ABCA4 cDNA expressed in the pCMV6-AC-GFP vector was purchased from OriGene Technologies. Two ABCA4 mutations, A1038V and G1961E, were generated using a QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies] [5 ]. Flp-In human embryonic kidney (HEK] 293 cells (Catalog#CRL-1573, Life Technologies] were cultured in Dulbecco’s minimal essential medium (DMEM) (Life Technologies] containing 10% fetal bovine serum (FBS), with penicillin (100 U/ml], streptomycin (100 μg/ml), and Zeocin (100 μg/mL) at 37°C in 5% CO2. The stable cell lines were generated as described previously [5 ]. We would state that all experiments were conducted with GFP tagged ABCA4. In our past experiments tagged CFTR behaved similarly to untagged versions [11 (link)–13 (link)].
Protocol detail
ABCA4 Mutant Expression in HEK Cells
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Cdna
Cell lines
Cells
Cftr
Cultured medium
Embryonic
Fetal bovine serum
Human
Kidney
Mutations
Penicillin
Site directed mutagenesis
Streptomycin
Vector
Zeocin
Corresponding Organization :
Other organizations : Johns Hopkins Medicine, Johns Hopkins University
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Variable analysis
independent variables
- A1038V ABCA4 mutation
- G1961E ABCA4 mutation
- ABCA4 expression and behavior
- Flp-In human embryonic kidney (HEK) 293 cells
- Dulbecco's minimal essential medium (DMEM) containing 10% fetal bovine serum (FBS), with penicillin (100 U/ml), streptomycin (100 μg/ml), and Zeocin (100 μg/mL)
- Temperature (37°C)
- CO2 level (5%)
- GFP tagged ABCA4 (positive control)
- Untagged CFTR (positive control)
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