Cloning and Expressing REEP6 Splice Variants

The REEP6 splice variant open reading frames were amplified from control D124 optic cup cDNA¹⁶ (link) using the primers REEP6_F1, 5′-GCTAGCCACCATGGACGGCCTGAGGCAGCGCGTGGAG-3′, and REEP6_R1stop, 5′-AATCTAGAGCGGCCGCTCACTTGTCCTTCGGCTGCGGGGTCTGGC-3′. The two observed PCR amplicons corresponded to the predicted REEP6.1 and REEP6.2 sizes and were excised and gel purified (QIAquick Gel Extraction Kit, QIAGEN) prior to cloning into the pSC-B-amp/kan vector (StrataClone Blunt PCR Cloning Kit, Agilent Technologies). Clone identities (REEP6.1, GenBank: NM_001329556.1; REEP6.2, GenBank: NM_138393.2) were confirmed by Sanger sequencing (Source BioScience). Mutations were introduced by site-directed mutagenesis (Q5 Site-Directed Mutagenesis Kit, New England Biolabs) using primers and conditions specified by the NEBaseChanger software. Sequence integrity was confirmed by Sanger sequencing as before. To create expression vectors, wild-type (WT) and mutant REEP6.1 sequences were cloned into pEYFP-N1 (Clontech) digested with BmtI and NotI to release the EYFP sequence. WT and mutant REEP6.1 expression plasmids were transfected into SK-N-SH (ATCC) cells using TransIT-LT1 Transfection Reagent (Mirus Bio) using the manufacturers’ recommended conditions, in 8-well Nunc Lab-Tek Permanox chamber slides (ThermoFisher Scientific) plated at 40,000 cells/well and 6-well plates plated at 500,000 cells/well.

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Arno G., Agrawal S.A., Eblimit A., Bellingham J., Xu M., Wang F., Chakarova C., Parfitt D.A., Lane A., Burgoyne T., Hull S., Carss K.J., Fiorentino A., Hayes M.J., Munro P.M., Nicols R., Pontikos N., Holder G.E., Asomugha C., Raymond F.L., Moore A.T., Plagnol V., Michaelides M., Hardcastle A.J., Li Y., Cukras C., Webster A.R., Cheetham M.E, & Chen R. (2016). Mutations in REEP6 Cause Autosomal-Recessive Retinitis Pigmentosa. American Journal of Human Genetics, 99(6), 1305-1315.

Publication 2016

QIAquick Gel Extraction Kit StrataClone Blunt PCR Cloning Kit Q5 Site-Directed Mutagenesis Kit pEYFP-N1 TransIT-LT1 Transfection Reagent Nunc Lab-Tek Permanox chamber slides

Cells Kan amp Mutations Open reading frames Optic Plasmids Primers Site directed mutagenesis Transfection Vectors

Corresponding Organization : Baylor College of Medicine

Other organizations : Moorfields Eye Hospital, NHS Blood and Transplant, University of Cambridge, Cambridge University Hospitals NHS Foundation Trust, Koret Foundation, National Eye Institute

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Protocol cited in 2 other protocols

Variable analysis

independent variables

Site-directed mutagenesis to introduce mutations in REEP6.1

dependent variables

Expression of wild-type (WT) and mutant REEP6.1 in SK-N-SH cells

control variables

Control D124 optic cup cDNA used as starting material
Transfection of WT and mutant REEP6.1 expression plasmids into SK-N-SH cells

controls

Positive control: wild-type (WT) REEP6.1 expression
Negative control: not explicitly mentioned

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