Gene Knockdown (KD) Strategy
Among the five pillar strategies proposed by the International Working Group for Antibody Validation (IWGAV), genetic strategy including gene knockout (KO) and knockdown (KD) is considered the best approach for validating antibody specificity. The underlying principle behind the genetic strategy is that if an antibody recognizes a signal in a wild-type (WT) cell or its lysate, but the signal disappears or significantly decreases in the KO/KD cell or its lysate, the antibody is considered to be specific.
In the past decade, KO approach has been used to target genes to validate antibody specificity. But this approach is not without disadvantages. It is estimated that one-fourth of human genes are essential. When these genes are completely deleted, cells will die or stop growing, making it impossible to obtain materials for antibody verification. In contrast, KD functions at the mRNA level to down-regulate protein expression. If designed properly, KD can achieve protein down-regulation at different levels. Such a feature makes KD particularly attractive for essential genes whose ablation (i.e. KO) is lethal. In laboratory, two approaches are commonly used for gene KD, namely small interfering RNA (siRNA) and small hairpin RNAs (shRNA). SiRNA-mediated RNA interference is transient and the KD effect varies greatly from experiment to experiment. On the other hand, lentiviruses can integrate shRNAs into a cell's genome, achieving a stable KD cell line. However, lentivirus-mediated shRNA strategy can be laborious and take 4-6 months, possibly longer if the first trial fails.
Our proprietary ShGE™ gene silencing platform uses lentiviruses to integrate specific shRNAs into cells' genome to stably knock down target genes. This allows us to achieve high throughput, low-cost validation of antibody specificity. These validated lentiviruses and cell lysates derived from our antibody validation process can also be used to effectively knock down your genes of interest and validate custom antibodies generated in your lab.