Our service includes custom shRNA construct preparation targeting any gene of interest.shRNA constructs are comprised of several options for client choice:
selection markers (fluorescent GFP or other color, antibiotic resistance)
inducible or constitutive promoter (U6 or H1)
We will design your target gene of interest in a standard shRNA vector or you can choose a custom design based on your needs.
All target gene constructs are cloned and verified before packaging into LentiPrep lentivirus particles. Validation of construct knockdown is also available to ensure rapid, guaranteed effect.
However transcript knockdown does not always correlate with phenotypic effect (our technical scientists can guide clients through this selection process).
Similar services are provided for shRNA and CRISPR knockdown approaches. Data packets for each are available on request.
(A) HEK293 cells (DIC) were transduced with LentiPrep particles expressing lacZ gene, and co-transduced with either LentiPrep particles containing shRNA to lacZ (lacZ), negative control lamin (lamin) gene, or mock transduced (Mock), with all vectors containing a co-expressed GFP marker (GFP) on the plasmid for transduction confirmation. (B) B-galactosidase activity assay to determine extent of shRNA knockdown of lacZ expression in cells from panel (A). B-galactosidase activity was normalized to control cells containing lacZ transduction alone, while shRNA co-transduced cells showed decreased lacZ expression depending on the gene targeted. This is in contrast to control cells with no lacZ expression which show no significant level of activity (n=3-5 experiments per treatment). (C) Western blot for lamin (shRNA knockdown) or actin (control, empty shRNA vector) proteins after stable transduction and expression of shRNA LentiPrep contructs. Each cell population (#1-5, bottom) were tested for lamin (lam) and lacZ (lac) expression, and all showed significant knockdown of lamin proteins (antibody recognizes lamins A/C as two separate sized bands at 68-78kD). Marker (M) shown on left side with sized bands.
Lentiviral vectors have become very useful tools for transgene delivery. Based on their ability to transduce both dividing and nondividing cells and to produce long-term transgene expression, lentiviruses have found numerous applications in the biomedical sciences, including developmental neuroscience.
Finally, we describe the infection of primary neuronal cultures with lentiviral vectors resulting in 85-90 % cell transduction using appropriate multiplicities of infection.
LentiPrep targeted shRNA against Tau can efficiently knockdown Tau protein (red) after 72 hours. DAPI stained nuclei (blue) identify cell numbers after 6 DIV.