Cloning Gene Variants and Reporter Assays.
Recent advances have identified new genetic markers associated with the inheritance of celiac disease. These non-HLA target regions remain to be fully categorized. Investigation of associated SNPs indicates that the causal variants may alter specific gene expression. Thus, closer examination of potential causal variants found within regulatory regions could provide data relating to the mechanistic association. Molecular cloning is an established fundamental tool that enables investigators to examine the differential potential at a variant site. In conjunction with reporter gene assays, SNPs affecting gene expression can be uncovered and contribute to our understanding of the underlying pathogenic mechanisms. This chapter outlines the protocols necessary to clone risk variants and transfect these constructs into a T cell line for reporter assay analysis.
Department of Medicine, Institute of Molecular Medicine, Trinity College Dublin, St. James's Hospital, Dublin 8, Ireland.
Ben
B
Molloy
Department of Medicine, Institute of Molecular Medicine, Trinity College Dublin, St. James's Hospital, Dublin 8, Ireland. rmcmanus@tcd.ie.
Ross
R
McManus
D002446:Celiac Disease / Q000235:genetics; D003001:Cloning, Molecular; D017930:Genes, Reporter; D006680:HLA Antigens / Q000235:genetics; D006801:Humans; D020641:Polymorphism, Single Nucleotide; D060888:Real-Time Polymerase Chain Reaction
D006680:HLA Antigens
Cloning; Luciferase; SNP; T cell lines