Adeno-associated Virus (AAV) Packaging

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Efficient transfection of viruses
Ubigene provides lentivirus, adenovirus and adeno-associated virus (AAV) with different sizes and grades of purification. Our products can easily achieve knockout (KO), overexpression and knockdown in vitro and in vivo.
Comparison of different viruses:
Type Lentivirus Adenovirus AAV
Origin HIV-1 Adenovirus type 5 AAV2
Genome Two linear and continuous single-stranded RNAs A linear and continuous double-stranded DNA A linear and continuous single-stranded DNA
Genome size ~9.2 kb (WT) ~36 kb (WT) ~4.7 kb (WT)
Expression Duration long-term stable expression Transient expression Long-term expression
Integrate to cell genome Yes No Low frequency
Immunogenicity Low High Very low
Capacity ~6.4 kb ~8.3 kb ~4.7 kb
Advantages

· Wide range of applications;
· High transduction efficiency;
· stable hereditary.

· High transduction efficiency;
· Large capacity;
· Expandable.

· Low immunogenicity;
· High safety;
· High specificity.

AAV Packaging Technical Detail
Adeno-associated virus (AAV) is a kind of Parvovirus, which genome is single-stranded DNA. It can infect both divisive and non-divisive cells. Adenoviruses or herpes viruses are usually needed to assist AVV to replicate in vivo. Recombinant AAV (rAAV) is a viral vector that combines the AAV2 genome with capsid protein genomes of different serotypes. CDS or RNAi interference sequences of the target genes can be inserted into the rAAV plasmid. AAV is mainly used in vivo injection because it has low immunogenicity and long-term expression.
Ubigene's AAV will be purified by ultracentrifugation, and its genome copy will be titrated by qPCR. The titer of AAV ranged from 10^12 to 10^13 v. g. / ml.
Different serotypes of AAVs have different transduction efficiency for tissues and cells. There are 10 different AAV serotypes in our packaging service. It is highly recommended that customers should select the corresponding AAV serotypes for different tissues:
Serotype Tissues
AAV1 SM,CNS,lung,retina,pancreas,heart,liver
AAV2 SM,SNS,liver,kidney,retina
AAV4 CNS,retina,lung,kidney
AAV5 SM,CNS,lung,retina
AAV6 SM,heart,lung,adipose,liver
AAV7 SM,retina,CNS,liver
AAV8 SM,CNS,retina,liver,pancreas,heart,kidney,adipose
AAV9 SM,lung,liver,heart,pancreas,CNS,retina,testes,kidney
AAVrh10 SM,lung,liver,heart,pancreas,CNS,retina,kidney
AAVPHP.B CNS
AAVDJ/8 SM, liver, CNS, retina
AAVPHP.S PNS
AAVPHP.eB CNS(stronger than AAVPHP.B)
Product Specs
Specs Titer Turnaround Application
Standard Size 1*10^11 GC 2-3weeks Cell transduction
Ultra-purified large size 1*10^12 GC 2-3weeks In vivo injection
Work Flow and Validation:


Knockout AAV
The gene knockout AAV vector would be constructed and packaged as AAV. After ultracentrifugation purification, it can be used for in vivo injection. Cells infected with AAV can stably express gRNA and Cas9 protein to knockout the target gene.
Gene knockout of AAV vector selection
YKO series Vector Reporter gene; Selection marker
AAV YKO plasmid YKO-AAV001-gRNA EGFP
YKO-AAV002-gRNA mCherry
Due to the limited capacity of AAV, SaCas9 protein can be used. SaCas9 and gRNA on the same construct can increase transduction efficiency.
Expression AAV
The gene overexpression vector would be constructed and packaged as AAV. After ultracentrifugation purification, it can be used for in vivo injection.
YOE series Vector Reporter gene; Selection marker
AAV YOE plasmid YOE-AAV001 EGFP
YOE-AAV002 mCherry
Customers can choose a variety of tissue-specific promoters that can achieve tissue-specific expression in vivo,Click to showshouzhi
Promoter Description Tissue Cell type Reference
Nes Rat nestin intron 2 enhancer fused to mouse Hsp68 minimal promoter Nervous system at embryonic stage Neural stem/pro-genitor cells Eur J Neurosci. 9:452 (1997); J Neurosci Res. 59:321 (2000)
Camk2a_short Mouse α-calcium-calmodulin dep-endent kinase II promoter (1.3 kb) Brain Pyramidal neurons Neuroscience. 161:441 (2009)
SYN1 Human synapsin I promoter Brain Mature neurons Mol Ther. 5:509 (2002)
Nes Rat neuron-specific enolase promoter Nervous system Various neurons Neuron. 5:187 (1990); J Mol Neurosci. 8:63(1997)
GFAP_short Human glial fibrillary acidic protein promoter (0.68 kb) Brain Astrocytes Glia. 56:481 (2008)
K14 Human keratin 14 promoter Epidermis Keratinocytes Gene. 153:297 (1995)
cTnT Chicken cardiac troponin T promoter Heart Cardiomyocytes Am J Physiol Cell Physiol. 286:C556 (2004); Gene Ther. 18: 43 (2011); Circ Res. 115:354 (2014)
alphaM-HC_short Mouse α-cardiac myosin heavy chain promoter (2.8 kb) Heart Cardiomyocytes Neuroscience. 161:441 (2009)
Myog Mouse myogenin promoter Muscle Myoblasts Genes Dev. 7:1277 (1993)
MHCK7 Composed of enhancer/promoter regions of murine muscle creatine kinase (MCK) and enhancer region of α-myosin heavy-chain genes. Genes Dev. 7:1277 (1993) Differentiated postmitotic stria-ted muscle cells Mol Ther. 15:320 (2007); Sci Transl Med. 9(418) (2017)
SM22a Mouse transgelin (a.k.a. Sm22a) promoter Adult smooth muscle Vascular smooth muscle cells J Cell Biol. 132:849 (1996)
EnSM22a The chimeric vascular smooth muscle-specific enhancer/promoter (The rabbit myosin heavy-chain enh- ancer inserted upstream of a truncated mouse SM22a promoter) Adult smooth muscle Vascular smooth muscle cells J Pharmacol Exp Ther. 329: 775 (2009)
Tie1 Mouse tyrosine kinase with immunoglobulin-like and EGF-like domains 1 promoter Blood vessel Endothelial cells J Cell Sci. 115:2075 (2002)
CD68_short Human CD68 promoter, short, recommended for in vitro use Blood Monocytes and macrophages Gene Ther. 19:1041 (2012)
Alb Mouse albumin promoter Liver Mature hepatocytes Genes Dev. 1:268 (1987)
TBG Human thyroxine-binding globulin promoter Liver Hepatocytes Gene. 506:289 (2012)
MMTV Mouse mammary tumor virus long terminal repeat (LTR) Mammary gland Ductal cells of the salivary gland, mammary epithelial cells Nucleic Acids Res. 25:4323 (1997)
HIP Human insulin promoter Pancreas β cells Cell. 52:773 (1998); Proc Natl Acad Sci U S A. 100: 6688 (2003)
Pdx1 Mouse pancreatic and duodenal homeobox 1 promoter Embryonic develo-ping pancreas and adult pancreatic islets Pancreatic proge-nitor cells and pancreatic β cells Development. 129:2447 (2002)
SP-B Human surfactant protein B promoter Lung AT II cells (alveolar type II epithelial cells) and Clara cells (bronchiolar epithelial cells) J Biol Chem. 270:24852 (1995); Gene Ther. 14:1461 (2007)
CD144 Human cadherin 5 (a.k.a. VE-Cadherin) promoter Lung, heart, ovary, spleen and kidney glomeruli Vascular endothelial cells Oncogene. 24:2992 (2005)
Knockdown AAV
The gene knockdown vector would be constructed and packaged as AAV. After ultracentrifugation purification, it can be used for in vivo injection.
YSH series Vector Reporter gene; Selection marker
AAV YSH plasmid YSH-AAV001-shRNA EGFP
YSH-AAV002-shRNA mCherry
Customers can choose a variety of tissue-specific promoters that can achieve tissue-specific expression in vivo.