Streamlined approach to functional analysis of promoter-region polymorphisms.
We have developed a rapid method for identifying functional promoter-region polymorphisms. Using a modified pGL3 luciferase expression T-vector, we can amplify by PCR, clone, identify allelic pairs of a polymorphic gene promoter region, and prepare plasmids for cell culture 10 promoters (20 allele pairs) per week per researcher. By utilizing 96-well plate technology and an internal control plasmid expressing secreted alkaline phosphatase, each of these allele pairs can be tested for relative promoter activity in each of three cell lines (HEK293t, TE671, and JEG3) with similar resources.
Coleman
S L
SL
University of Wales College of Medicine, Cardiff, UK.
Hoogendoorn
B
B
Guy
C
C
Smith
S K
SK
O'Donovan
M C
MC
Buckland
P R
PR
D000483:Alleles; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology; D008527:Medulloblastoma / Q000235:genetics; D010920:Placenta / Q000166:cytology; D011110:Polymorphism, Genetic / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011786:Quality Control; D012680:Sensitivity and Specificity