Delivery of short hairpin RNA sequences by using a replication-competent avian retroviral vector

Abstract

While recent studies have demonstrated that retroviral vectors can be used to stably express short hairpin RNA (shRNA) to inhibit gene expression, these studies have utilized replication-defective retroviruses. We describe the creation of a replication-competent, Gateway-compatible retroviral vector capable of expressing shRNA that inhibits the expression of specific genes.

FIG. 1.

Schematic representation of a Gateway-compatible, replication-competent, retroviral vector capable of delivering shRNA. (A) Gateway-compatible RCANBP(A). (B) The RCANBP-H1-shRNA viral vector has been engineered to express shRNA by introducing an RNA Pol III-shRNA cassette, containing the H1 promoter upstream of the shRNA stem-loop sequence. SA, splice acceptor; SD, splice donor; LTR, long terminal repeat; ψ, packaging signal.

FIG. 2.

Ability of RCANBP-H1-GAPDH to express GAPDH shRNA and to reduce GAPDH expression in A375-TVA cells. (A) Polyacrylamide gel electrophoresis-Northern analysis of GAPDH shRNA expression using the sense strand as an RNA probe. A375-TVA cells were uninfected (− [control]) or were infected with RCANBP-H1-GAPDH for one (1x) or two (2x) rounds. Samples were normalized for total RNA content, and 70 μg of total cellular RNA was analyzed per sample. On the sense strand, GAPDH shRNA (19 nucleotides [nt]) was used at 100, 10, and 1 pmol as a size marker and for quantitation of shRNA production. The upper band likely corresponds to hairpin-loop formation (47 nt) of the shRNA prior to loop digestion. (B) Northern blot analysis of GAPDH message in A375-TVA cells that were uninfected (lane 1) or infected with RCANBP-H1-GAPDH (lane 2) for one round. Blots were stripped and reprobed with 18S RNA to ensure equal loading of RNA.