gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
Adenovirus shRNA Knockdown Vector
The Adenovirus shRNA Knockdown vector system is an efficient method for stably knocking down expression of a target gene in many (but not all) mammalian cell types. The vector remains as episomal DNA in cells, without integrating into the host genome, and is often the preferred vector system for in vivo use.
Adenoviral vectors are derived from adenovirus, a double-stranded linear DNA virus which causes the common cold.
An adenoviral vector is first constructed as a plasmid in E. coli, and is then transfected into packaging cells, where the region of the vector between the two inverted terminal repeats (ITRs) is packaged into live virus.
After the viral genome is delivered into target cells, it enters the nucleus and remains as episomal DNA. The shRNA is stably expressed from a human U6 promoter, leading to degradation of target gene mRNA within infected cells.
By design, our adenoviral vectors lack the E1A, E1B and E3 genes (delta E1 + delta E3). The first two are required for the production of live virus (these two genes are engineered into the genome of packaging cells). As a result, virus produced from the vectors have the important safety feature of being replication incompetent (meaning that they can transduce target cells but cannot replicate in them).
For further information about this vector system, please refer to the papers below.
|Proc Natl Acad Sci U S A. 91:8802 (1994)||The 2nd generation adenovirus vectors|
|J Gen Virol. 36:59 (1977)||A packaging cell line for adenovirus vectors|
|J Virol. 79:5437 (2005)||Replication-competent adenovirus (RCA) formation in 293 Cells|
|Gene Ther. 3:75 (1996)||A cell line for testing RCA|
Our vector is derived from the adenovirus serotype 5 (Ad5). It is optimized for high copy number replication in E. coli, high-titer packaging of live virus, efficient transduction of host cells, and effective gene knockdown. The human U6 promoter drives high-level, constitutive transcription of the shRNA in mammalian cells, while our optimized shRNA stem-loop sequences mediate efficient shRNA processing and target gene knockdown.
Low risk of host genome disruption: Upon transduction into host cells, adenoviral vectors remain as episomal DNA in the nucleus. The lack of integration into the host genome can be a desirable feature for in vivo human applications, as it reduces the risk of host genome disruption that might lead to cancer.
Very high viral titer: After our adenoviral vector is transfected into packaging cells to produce live virus, the virus can be further amplified to very high titer by re-infecting packaging cells. This is unlike lentivirus, MMLV retrovirus, or AAV, which cannot be amplified by re-infection. When adenovirus is obtained through our virus packaging service, titer can reach >1010 plaque-forming unit per ml (PFU/ml).
Broad tropism: Cells from commonly used mammalian species such as human, mouse and rat can be transduced with our adenoviral vectors. But some cell types have proven difficult to transduce (see disadvantages below).
Effectiveness in vitro and in vivo: Our vector is often used to transduce cells in live animals, but it can also be used effectively in vitro.
Safety: The safety of our vector is ensured by the fact that it lacks genes essential for virus production (these genes are engineered into the genome of packaging cells). Virus made from our vector is therefore replication incompetent except when it is used to transduce packaging cells.
Non-integration of vector DNA: The adenoviral genome does not integrate into the genome of transduced cells. Rather, it exists as episomal DNA, which can be lost over time, especially in dividing cells.
Difficulty transducing certain cell types: While our adenoviral vectors can transduce many different cell types including non-dividing cells, it is inefficient against certain cell types such as endothelia, smooth muscle, differentiated airway epithelia, peripheral blood cells, neurons, and hematopoietic cells.
Strong immunogenicity: Live virus from adenoviral vectors can elicit strong immune response in animals, thus limiting certain in vivo applications.
Technical complexity: The use of viral vectors requires the production of live virus in packaging cells followed by the measurement of viral titer. These procedures are technical demanding and time consuming relative to conventional plasmid transfection. These demands can be alleviated by choosing our virus packaging services when ordering your vector.
5' ITR: 5' inverted terminal repeat. In wild type virus, 5' ITR and 3' ITR are essentially identical in sequence. They reside on two ends of the viral genome pointing in opposite directions, where they serve as the origin of viral genome replication.
Ψ: Adenovirus packaging signal required for the packaging of viral DNA into virus.
U6 Promoter: Drives expression of the shRNA. This is the promoter of the human U6 snRNA gene, an RNA polymerase III promoter which efficiently expresses short RNAs.
Sense, Antisense: These sequences are derived from your target sequences, and are transcribed to form the stem portion of the “hairpin” structure of the shRNA.
Loop: This optimized sequence is transcribed to form the loop portion of the shRNA “hairpin” structure.
Terminator: Terminates transcription of the shRNA.
hPGK promoter: Human phosphoglycerate kinase 1 gene promoter. It drives the ubiquitous expression of the downstream marker gene.
Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.
SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.
ΔAd5: Portion of Ad5 genome between the two ITRs minus the E1A, E1B and E3 regions.
3' ITR: 3' inverted terminal repeat. See description for 5’ ITR.
pBR322 ori: pBR322 origin of replication. Plasmids carrying this origin exist in medium copy numbers in E. coli.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
PacI: PacI restriction site (PacI is a rare cutter that cuts at TTAATTAA). The two PacI restriction sites on the vector can be used to linearize the vector and remove the vector backbone from the viral sequence, which is necessary for efficient packaging.