A lentiviral system for efficient knockdown of proteins in neuronal cultures [version 1; referees: 2 approved]

Abstract

We have devised a protocol for highly efficient and specific knockdown of proteins in neuronal cultures. Small hairpin RNAs (shRNAs) are embedded into a microRNA (miRNA) context by oligo annealing to create shRNAmiRs, which are expressed from within the 3'-UTR of a reporter protein. This reporter protein/synthetic miRNA cassette is transferred to a targeting vector and lentivirus is produced in HEK-293-T cells following co-transfection of the targeting vector with three additional vectors encoding essential lentiviral proteins. Mature virus is harvested by collecting culture medium from transfected HEK-293-T cells, the virus is purified by centrifugation, and virus titers are determined prior to addition to neuronal cultures. Near 100% transduction efficiency of cultured hippocampal neurons is routinely observed and allows for the population-wide inhibition of target protein expression and the simultaneous knockdown of multiple proteins with little or no toxicity. The lentivirus generated can be used for protein knockdown in multiple neuronal culture models and at a variety of developmental stages. The steps from shRNAmiR design to ready-to-use virus stocks can be completed in as little as two weeks.

Figure 1. Lentiviral-mediated expression of shRNAmiR knockdown cassettes allows for efficient protein knockdown in primary hippocampal neurons

(A–C) Western blot analysis of total protein extracts from rat primary hippocampal neurons for various proteins as indicated. Clathrin heavy chain (CHC) was used as loading control. dyn 1: dynamin 1, α-A: α-adaptinA, α-C: α-adaptinC, control: shRNAmiR-control virus. (A) Primary hippocampal neurons were transduced on DIV 4 with different shRNAmiR viruses directed against dynamin 1 as indicated by the nt positions and a range of MOIs were tested for each virus. Cells were harvested for analysis on DIV 10. The generation of four to five different knockdown viruses against one target protein usually yields in at least two viruses with high knockdown efficacy. (B) Primary hippocampal neurons were transduced on DIV 4 (left) or DIV 14 (right) with different shRNAmiR viruses and directed against dynamin 1 and MOIs as indicated and Western blot for dynamin 1 confirms efficient protein knockdown in neurons transduced as different stages of in vitro differentiation. The dynamin 1 blot on the left also demonstrated the efficacy of the KD. Dynamin 1 is virtually undetectable in knockdown neurons after ECL exposure for two minutes using regular ECL and faint traces of residual dynamin 1 are only detected under conditions that super-saturate the signal detected in shRNAmiR-control neurons (10 min super-ECL). (C) Knockdown of additional proteins involved in clathrin-mediated endocytosis as indicated. Neurons were transduced at DIV 4 and harvested for analysis on DIV 28, demonstrating the persistent knockdown of target proteins over extended periods of time. (D) Electron microscopy analysis of mouse primary cortical neurons transduced with control and dynamin 1 knockdown shRNAmiR viruses as indicated. The lentivirus-mediated knockdown recapitulates the phenotype seen in neurons from dynamin 1 knockout animals and arrow highlight the abundance of clathrin-coated pits that accumulate due to dynamin 1 depletion.

Figure 2. Immunofluorescence analysis of dynamin 1 expression in control and dynamin 1 knockdown neurons

Primary rat hippocampal neurons were transduced on DIV 4 with control or dynamin 1 knockdown shRNAmiR viruses as indicated using an MOI of 12 and processed for immunofluorescence on DIV 21. Two examples for each condition are shown. GFP is the fluorescent marker protein expressed as part of the shRNAmiR cassette and dynamin 1 was detected using an antibody directed against endogenous dynamin 1. Dynamin 1 is readily detected in neurons transduced with the control virus but is virtually undetectable in neurons transduced with the knockdown viruses. The areas in the white boxes seen in the lower magnification images is shown at higher magnification below each image.