Full-length 3′-untranslated region reporter construction with recombineering

Abstract

Complexity in higher animals derives in part from various modalities of protein-coding gene expression regulation, including microRNA repression by binding to 3′-untranslated regions (UTRs) of specific genes. Reporter constructs containing candidate microRNA target sites are a popular approach of functional studies, and full-length 3′-UTR sequences are preferred because they contain all regulatory elements and preserve higher order structure as much as possible. However, this approach is often handicapped by the extreme length of the 3′-UTR. Here, we present a rapid and accurate cloning procedure to generate full-length 3′-UTR reporter constructs by recombinogenic engineering (recombineering) in vivo cloning. The approach includes making retrieval constructs by sequence- and ligation-independent cloning (SLIC) and retrieving the full-length 3′-UTR in one exon to the retrieval construct from a bacterial artificial chromosome (BAC) by recombineering to generate the final full-length 3′-UTR reporter construct for the gene of interest. This method is successfully implemented with mouse full-length 3′-UTRs of Igf1 (6.5kb), Igf1r (7.5kb), and Sp1 (5.5kb). Expansion of this method is adaptable to retrieve 3′-UTRs encoded in more than one exon by removing the introns from the BAC first with recombineering. This method will advance functional studies of regulation of gene expression at the post-transcriptional level through microRNA suppression.