Adenovirus ADV-He Yuan Li Ji

Adenovirus ADV

Original link:Harmony with Metaorganisms - Adenovirus ADV

Adenovirus (ADV) is a linear double stranded DNA virus without an envelope, with a wide range of cell and tissue infectivity. The infection process is intense, making it suitable for experiments with high gene expression in a short period of time. Compared with other viruses, adenoviruses have the advantage of longer insertion fragments and stronger expression activity.

　　When used in vitro, adenovirus vectors have high transgenic efficiency (close to 100% transduction efficiency) and can transduce different types of cells, making them a good tool for delivering genes to a large number of difficult to transfect cells. However, when applied in vivo, due to the strong immunogenicity and severe infection of adenovirus, it often causes local tissue inflammation and immune response in animals, which affects the objectivity of animal signs and experimental results.

ADV packaging and infection process

Packaging and purification of adenovirus

　　The adenovirus packaging skeleton plasmid and shuttle plasmid were co transfected into HEK-293 cells. After the cells showed CPE state, the virus was purified by collecting cell supernatant and lysate, and then concentrated or centrifuged with cesium chloride density gradient.

　　CPE（Cytopathic Effect）：The cellular degeneration caused by virus infection on tissue cultured cells can be quantified using this pathological effect.

Adenovirus titer detection

　　Detection method: Enzyme linked immunosorbent assay (ELISA) for adenovirus titer measurement. The virus titer is calculated based on the number of positive cells stained brown after infection.

Experimental principle: The capsid protein expressed in positive cells infected with adenovirus is stained brown.

Detection method: Immunocolorimetric method

Experimental principle: The monoclonal antibody against the coat protein of type 5 adenovirus binds to the cells of the adenovirus sample to be infected, and then the HRP labeled secondary antibody binds to the primary antibody. The infected cells turn brown after staining with the colorimetric solution. The viral titer is calculated based on the number of positive cells.

Advantages of adenovirus vectors

① Express quickly, after adenovirus infection of cells, it can be expressed within 1-2 days

② Large carrier capacity, high infection efficiency, commonly used to infect cells that are difficult to infect

③ Not integrated into chromosomes, no insertion mutagenicity

④ It has hepatotropism and is prone to infecting liver cells. The in vivo use of adenoviruses is for infecting the liver

　 However, due to the high immunogenicity of adenoviruses, they are prone to inflammation in certain animal models and tissues that are sensitive to external stimuli, and caution should be exercised when used in vivo.

Comparison of Biological Characteristics of Different Viruses

Carrier selection

　　Heyuan Biotechnology has a rich range of adenovirus products, which are used to manipulate coding and non coding genes, such as lncRNA microRNA、circRNA。

The following adenovirus vectors are available for you to choose from:

Regulatory methods	Carrier number	Carrier name (order of carrier components)	Eukaryotic resistance	fluorescence	Promoter	Default label	Carrier capacity
Overexpression	H225	pADV-mCMV-MCS-3xFLAG	N/A	N/A	mCMV	3FLAG	>6kb
	H201	pADV-mCMV-EGFP-3xFLAG	N/A	EGFP	mCMV	3FLAG	>5kb
	H213	pADV-mCMV-3xFLAG-EGFP	N/A	EGFP	mCMV	3FLAG	>5kb
	H221	pADV-mCMV-3xFLAG-P2A-EGFP	N/A	EGFP	mCMV	3FLAG	>5kb
	H204	pADV-mCMV-mCherry-HA	N/A	mCherry	mCMV	HA	>5kb
	H222	pADV-mCMV-3xFLAG-T2A-mCherry	N/A	mCherry	mCMV	3FLAG	>5kb
	H12588	pADV-EF1-mNeonGreen-CMV-MCS-3xFLAG	N/A	mNeonGreen	CMV	3FLAG	>4kb
	H12589	pADV-EF1-mScarlet-CMV-MCS-3xFLAG	N/A	mScarlet	CMV	3FLAG	>4kb
CircRNA overexpression	H6946	pADV-CMV-S-circRNA	N/A	N/A	CMV	N/A	>5kb
MiRNA overexpression	H216	pADV-U6-miR30(miRNA)-CMV-EGFP	N/A	EGFP	U6	N/A	N/A
Expressing cre	K0024	pADV-CMV-Cre	N/A	N/A	CMV	N/A	N/A
interfere	DKD001	pADV-U6-shRNA-CMV-EGFP	N/A	EGFP	U6	N/A	N/A
interfere	DKD004	pADV-U6-shRNA-CMV-mCherry	N/A	mCherry	U6	N/A	N/A
CRISPR Knockout	H5064	pADV-U6-spgRNA v2.0-CMV-3xFLAG-spCas9	N/A	N/A	U6	3FLAG	N/A
	H9350	pADV-U6-spgRNA v2.0-CMV-sfGFP-P2A-3xFLAG-spCas9	N/A	sfGFP	U6	3FLAG	N/A
	H5066	pADV-U6-spgRNA v2.0-CMV-EGFP	N/A	EGFP	U6	N/A	N/A
	H218	pADV-CMV-mCherry-P2A-3xFLAG-spCas9	N/A	mCherry	CMV	3FLAG	N/A
	H5449	pADV-CMV-mCherry-P2A-3xFLAG-eSpCas9_1.1	N/A	mCherry	CMV	3FLAG	N/A

Application instance

　　1.Hepatology. (IF=14.079). Zhou YF,et.al. (2018). Cystathionine β-synthase is required for body iron homeostasis. [腺病毒, 肝脏]

　　Infection site: liver

　　Carrier name：Ad-CBS

　　Injection method: tail vein

　　Injection volume：1 x1011 PFU/mL，10ul

　　Testing time: 7 days

　　2. Diabetes. (IF=8.095). Xiao Y Z,et.al. (2015). Activation of ERK1/2 ameliorates liver steatosis in leptin receptor deficient (db/db)mice via stimulating ATG7-dependent autophagy. [Adenovirus, Liver]

　　Infection site: liver

　　Carrier name：Ad-ATG7

　　Injection method: tail vein

　　Injection volume：1 x109 PFU/piece

　　Testing time: 10 days

　　3. Molecular Neurobiology. (IF=5.397). Guo J,et.al. (2015). Overexpression of Fibulin-5 Attenuates Ischemia/Reperfusion Injury After Middle Cerebral Artery Occlusion in Rats. [Adenovirus, Rat]

　　Injection site: rat cerebral cortex

　　Carrier name：Ad-FBLN

　　Injection method: Brain stereotactic injection

　　Injection volume: three different doses

　　Testing time: 7 days

　　The production and quality control of adenovirus from Heyuan Biotechnology adopts internationally recognized standard processes, using AdEasy and Admax adenovirus packaging systems for packaging in HEK-293 cells. The obtained virus particles were subjected to ultrafast density gradient centrifugation and filtration, and their titers were determined by chitosan immunoassay. In general, the titer of adenovirus is between 1010~1011pfu/ml, which can fully meet the requirements of various experiments.