Adenoviral Vectors

Ad5 vectors are the most commonly used adenovirus vector. Ad5 vectors transduce a wide range of cell types. They utilize the Coxsackie-Adenovirus Receptor (CAR) to enter cells. Transduction efficiency is related to the level of CAR expression on the cell membrane. It your target cells has low level of CAR or do not express CAR, you may want to consider our chimeric virus, Ad5F35. In general, Ad5 vectors transduce human cells quite well, whereas mouse cells are mostly refractory to Ad5.

If you are working with an Ad5 vector and need to transduce low-or non-CAR expressing cells, we highly recommend incubating your vector with GeneJammer (Agilent) prior to transduction. The publication below shows an extensive set of experiments comparing the transduction efficiency of Ad5 into cells with low-CAR expression in the presence and absence of GeneJammer. Data show that GeneJammer dramatically increases the number of cells transduced. For more details, take a look at C.M. Fouletier-Dilling et al. 2005, Human Gene Therapy, 16:1287–1297.

Adenovirus 5 is an important vector because it can infect a variety of non-dividing cells and elicit high levels of transgene expression. However, it poorly transduces a number of human cells and tissues, mainly because they lack the Coxsackie adenovirus receptor (CAR). A new adenovirus vector has been developed where the fiber gene from adenovirus type 35 has been substituted for the Ad5 fiber. Ad35 fiber does not bind CAR and enters the cell by a different mechanism. Hematopoietic cells, primitive hematopoietic stem cells, and human skin fibroblasts are an example of cells that showed a higher transduction efficiency with Ad5F35 than their Ad5 counterparts.

RNA interference is a method of eliminating gene expression via post-transcriptional gene silencing. RNAi is activated by introducing double-stranded RNA homologous to the target gene transcript. The exogenous RNA is digested into small interfering RNAs (siRNA), which bind a nuclease complex to form an RNA-induced silencing complex. This complex targets the endogenous gene transcripts by base-pairing and cleaves the mRNA. This methodology is achieved both quickly and easily in both animal and cell-line models. The success of this methodology depends on choosing the proper target sequence within the gene of interest and proper design of the siRNA oligonucleotides. You will need a gene-specific assay to test for suppression of your gene of interest. This assay may be Western blot with an antibody to a protein encoded by your gene, RT-PCR using primers to your gene, Northern blot with a probe to your gene or some other functional assay for the protein encoded by your gene.