An infinitely expandable cloning strategy plus repeat-proof PCR for working with multiple shRNA

• Published in: PloS one Vol. 3; no. 11; p. e3827
• Main Authors: McIntyre, Glen John, Groneman, Jennifer Lynne, Tran, Anna, Applegate, Tanya Lynn
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 27-11-2008; Public Library of Science (PLoS)
• Subjects: Biotechnology; Cassettes; Cloning; Cloning vectors; Cloning, Molecular - methods; Computer simulation; Construction; Cores; Deoxyribonucleic acid; DNA; DNA Restriction Enzymes; DNA-Directed DNA Polymerase; E coli; Enzymes; Escherichia coli; Expression vectors; Fragmentation; Fragments; Gene expression; Growth factors; MicroRNAs; Molecular Biology/Post-Translational Regulation of Gene Expression; Plasmids; Polymerase chain reaction; Polymerase Chain Reaction - methods; Ribonucleic acid; RNA; RNA, Small Interfering - genetics; RNA-mediated interference; Sequences; Virology/New Therapies, including Antivirals and Immunotherapy
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0003827

Vector construction with restriction enzymes (REs) typically involves the ligation of a digested donor fragment (insert) to a reciprocally digested recipient fragment (vector backbone). Creating a suitable cloning plan becomes increasingly difficult for complex strategies requiring repeated insertions such as constructing multiple short hairpin RNA (shRNA) expression vectors for RNA interference (RNAi) studies. The problem lies in the reduced availability of suitable RE recognition sites with an increasing number of cloning events and or vector size. This report details a technically simple, directional cloning solution using REs with compatible cohesive ends that are repeatedly destroyed and simultaneously re-introduced with each round of cloning. Donor fragments can be made by PCR or sub-cloned from pre-existing vectors and inserted ad infinitum in any combination. The design incorporates several cloning cores in order to be compatible with as many donor sequences as possible. We show that joining sub-combinations made in parallel is more time-efficient than sequential construction (of one cassette at a time) for any combination of 4 or more insertions. Screening for the successful construction of combinations using Taq polymerase based PCR became increasingly difficult with increasing number of repeated sequence elements. A Pfu polymerase based PCR was developed and successfully used to amplify combinations of up to eleven consecutive hairpin expression cassettes. The identified PCR conditions can be beneficial to others working with multiple shRNA or other repeated sequences, and the infinitely expandable cloning strategy serves as a general solution applicable to many cloning scenarios.