AAV Serotype Testing Panel
Adeno-associated virus (AAV)는 다양한 포유류 세포 유형에 transduction할 수 있는 능력과 숙주 생물체에서 immunogenicity가 낮기 때문에 유전자 치료에 가장 효과적인 바이러스 벡터로 부각되고 있습니다. VectorBuilder는 AAV serotype testing panel을 제공하여 사용자가 세포 또는 동물의 다양한 serotypes을 체계적으로 비교하여 특정 용도에 적합한 최적의 AAV serotype을 선택할 수 있도록 하였습니다.
중점 사항
- VectorBuilder의 AAV serotype testing panel은 프로젝트 요구 사항에 따라 18개 serotype의 컬렉션에서 3개 이상의 AAV unit으로 AAV 패널을 맞춤화할 수 있는 유연성을 제공하는 AAV serotype testing 키트입니다.
- 또한 AAV panel을 주문할 때 AAV 매개 EGFP 리포터 발현을 구동하기 위해 CAG 또는 CMV promoter 중에서 선택할 수 있는 옵션이 있어 panel에서 다양한 serotype의 성능을 모니터링하고 비교할 수 있습니다.
주문 방법 Price Match
| Product Name | Catalog No. | Titer & Volume | Unit Price (USD) | Availability |
|---|---|---|---|---|
| In vitro grade AAV serotype testing panel (CMV-EGFP) | PANEL-AAVS01 | 10 12GC/ml, 25 ul | $79 per aliquot | In stock |
| In vitro grade AAV serotype testing panel (CAG-EGFP) | PANEL-AAVS02 | |||
| In vivo grade AAV serotype testing panel (CMV-EGFP) | PANEL-AAVSP01 | 10 13GC/ml, 25 ul | $159 per aliquot | |
| In vivo grade AAV serotype testing panel (CAG-EGFP) | PANEL-AAVSP02 |
Notes:
a. Our standard AAV serotype collection includes AAVs 1, 2, 3, 4, 5, 6, 6.2, 7, 8, 9, rh10, DJ, DJ/8, PHP.eB, PHP.S, AAV2-retro, AAV2-QuadYF and AAV2.7m8.
b. The AAVs packaged in this offering are ssAAVs. If you need scAAVs (self-complementary AAVs), please send a design requestto us.
c. 각 panel에는 serotype에 상관없이 최소 3개 이상의 AAV unit이 필요합니다 (예를 들어 AAV5 2 unit, AAV9 1 unit). Panel을 고객 맞춤형으로 제작하는 경우에는 디자인 의뢰하기 로 문의하여 주시기 바랍니다.
배송 및 보관
Non-ultra-purified AAV는 Tris-based buffer에 저장되고 ultra-purified AAV는 PBS-based buffer에 저장됩니다. 수령 시 장기 보관의 경우 -80°C에서 보관해야 하고(최소 1년 동안 안정), 예를 들어 2-3주 단기의 경우는 -20°C에 보관 가능합니다. 해동된 AAV는 4°C에서 1-2주 동안 보관할 수 있습니다.
기술적인 정보
AAV vector
AAV serotype testing panel에는 매우 최적화된 AAV 벡터 시스템을 사용하여 EGFP를 발현하는 premade AAV가 포함되어 있습니다. 사용자는 EGFP 발현을 유도하기 위해 CAG 또는 CMV promoter 중에서 선택할 수 있습니다. AAV 벡터 시스템은 E. coli에서 높은 카피 수 복제, 살아있는 바이러스의 고역가 패키징, 호스트 세포의 효율적인 transduction 및 높은 수준의 transgene 발현에 최적화되어 있습니다. AAV 벡터는 알려진 모든 capsid serotype을 사용하여 바이러스로 패키징될 수 있으며, 매우 높은 transduction 효율이 가능하며, 안전성 리스크가 낮습니다.
Figure 1.(A) CMV promoter 또는 (B) CAG promoter를 포함한 EGFP-발현 AAV vectors 맵
AAV serotypes의 조직 친화성
많은 AAV 종류가 자연에서 확인되었습니다. 이들은 바이러스 표면에 있는 capsid 단백질의 다른 항원성에 따라 serotype이 나뉩니다. 상이한 serotype은 다른 조직 친화성 (즉, 감염의 조직 특이성)을 갖는 바이러스를 생성할 수 있습니다. VectorBuilder의 AAV 벡터가 바이러스에 패키징될 때 패키징에 다른 capsid 단백질을 사용하여 다른 serotype을 바이러스에 부여할 수 있습니다. AAV serotype testing panel은 여러 AAV serotype의 조직 특이성을 체계적으로 비교하여 실험에 가장 적합한 serotype을 선택하도록 디자인되었습니다. 아래 표는 다양한 AAV serotype에 의해 부여된 조직 특이성을 나열한 것입니다.
List by Serotype
List by Tissue Type
| Serotype | Tissue tropism |
|---|---|
| AAV1 | Smooth muscle, skeletal muscle, CNS, brain, lung, retina, inner ear, pancreas, heart, liver |
| AAV2 | Smooth muscle, CNS, brain, liver, pancreas, kidney, retina, inner ear, testes |
| AAV3 | Smooth muscle, liver, lung |
| AAV4 | CNS, retina, lung, kidney, heart |
| AAV5 | Smooth muscle, CNS, brain, lung, retina, heart |
| AAV6 | Smooth muscle, heart, lung, pancreas, adipose, liver |
| AAV6.2 | Lung, liver, inner ear |
| AAV7 | Smooth muscle, retina, CNS, brain, liver |
| AAV8 | Smooth muscle, CNS, brain, retina, inner ear, liver, pancreas, heart, kidney, adipose |
| AAV9 | Smooth muscle, skeletal muscle, lung, liver, heart, pancreas, CNS, retina, inner ear, testes, kidney, adipose |
| AAV-rh10 | Smooth muscle, lung, liver, heart, pancreas, CNS, retina, kidney |
| AAV-DJ | Liver, heart, kidney, spleen |
| AAV-DJ/8 | Liver, brain, spleen, kidney |
| AAV-PHP.eB | CNS |
| AAV-PHP.S | PNS |
| AAV2-retro | Spinal nerves |
| AAV2-QuadYF | Endothelial cell, retina |
| AAV2.7m8 | Retina, inner ear |
| Tissue type | Recommended AAV serotypes |
|---|---|
| Smooth muscle | AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-rh10 |
| Skeletal muscle | AAV1, AAV9 |
| CNS | AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAV-rh10, AAV-PHP.eB |
| PNS | AAV-PHP.S |
| Brain | AAV1, AAV2, AAV5, AAV7, AAV8, AAV-DJ/8 |
| Retina | AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAV-rh10, AAV2-QuadYF, AAV2.7m8 |
| Inner ear | AAV1, AAV2, AAV6.2, AAV8, AAV9, AAV2.7m8 |
| Lung | AAV1, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV9, AAV-rh10 |
| Liver | AAV1, AAV2, AAV3, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV-rh10, AAV-DJ, AAV-DJ/8 |
| Pancreas | AAV1, AAV2, AAV6, AAV8, AAV9, AAV-rh10 |
| Heart | AAV1, AAV4, AAV5, AAV6, AAV8, AAV9, AAV-rh10, AAV-DJ |
| Kidney | AAV2, AAV4, AAV8, AAV9, AAV-rh10, AAV-DJ, AAV-DJ/8 |
| Adipose | AAV6, AAV8, AAV9 |
| Testes | AAV2, AAV9 |
| Spleen | AAV-DJ, AAV-DJ/8 |
| Spinal nerves | AAV2-retro |
| Endothelial cells | AAV2-QuadYF |
실험에 의한 검증
VectorBuilder는 triple transfection 기반 AAV 패키징 프로토콜을 최적화하기 위해 여러 독점 기술을 개발했으며 당사의 바이러스는 포유동물 세포에서 높은 transduction 효율을 나타내는 것으로 검증되었습니다.
Figure 2. HEK293T 세포는 동일한 CMV>EGFP 벡터 ( VB010000-9394npt)로 패키징된 18개 serotype의 재조합 AAV로 transduction 되었습니다. 각기 다른 serotype으로 transduction 후 48시간에 EGFP 발현을 확인하였습니다. Scale bars: 100 μm.
Figure 3.HeLa 세포는 동일한 CMV>EGFP 벡터 ( VB010000-9394npt)로 패키징된 18개 serotype의 재조합 AAV로 transduction 되었습니다. EGFP(A) Mean Fluorescence Intensity (MFI) 및 (B) 양성은 transduction 48시간 후 flow cytometry를 사용하여 정량화되었습니다. (C) 각기 다른 serotype으로 transduction 후 48시간에 EGFP 발현을 확인하였습니다. Scale bars: 100 μm.
Figure 4.Huh-7 세포는 동일한 CMV>EGFP 벡터 ( VB010000-9394npt)로 패키징된 18개 serotype의 재조합 AAV로 transduction 되었습니다. EGFP(A) Mean Fluorescence Intensity (MFI) 및 (B) 양성은 transduction 48시간 후 flow cytometry를 사용하여 정량화되었습니다. (C) 각기 다른 serotype으로 transduction 후 48시간에 EGFP 발현을 확인하였습니다. Scale bars: 100 μm.
자료
문서 자료
Certificate of Analysis (COA) Brochures & Flyers주요 인용 논문
Trb3 Controls Mesenchymal Stem Cell Lineage Fate and Enhances Bone Regeneration by Scaffold-Mediated Local Gene Delivery
Jiabing Fan, et al.
Biomaterials, 2020, doi: 10.1016/j.biomaterials.2020.120445
Products used:
- Adeno-Associated Virus (AAV) Packaging (AAV1, 2, 5, 6, 8 & 9)
- Lentivirus Packaging
- Vector Cloning
Abstract:Aberrant lineage commitment of mesenchymal stem cells (MSCs) in marrow contributes to abnormal bone formation due to reduced osteogenic and increased adipogenic potency. While several major transcriptional factors associated with lineage differentiation have been found during the last few decades, the molecular switch for MSC fate determination and its role in skeletal regeneration remains largely unknown, limiting creation of effective therapeutic approaches. Tribbles homolog 3 (Trb3), a member of tribbles family pseudokinases, is known to exert diverse roles in cellular differentiation. Here, we investigated the reciprocal role of Trb3 in the regulation of osteogenic and adipogenic differentiation of MSCs in the context of bone formation, and examined the mechanisms by which Trb3 controls the adipo-osteogenic balance. Trb3 promoted osteoblastic commitment of MSCs at the expense of adipocyte differentiation. Mechanistically, Trb3 regulated cell-fate choice of MSCs through BMP/Smad and Wnt/β-catenin signals. Importantly, in vivolocal delivery of Trb3 using a novel gelatin-conjugated caffeic acid-coated apatite/PLGA (GelCA-PLGA) scaffold stimulated robust bone regeneration and inhibited fat-filled cyst formation in rodent non-healing mandibular defect models. These findings demonstrate Trb3-based therapeutic strategies that favor osteoblastogenesis over adipogenesis for improved skeletal regeneration and future treatment of bone-loss disease. The distinctive approach implementing a scaffold-mediated local gene transfer may further broaden the translational use of targeting specific therapeutic gene related to lineage commitment for clinical bone treatment.
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Fructose-1, 6-bisphosphatase is a nonenzymatic safety valve that curtails AKT activation to prevent insulin hyperresponsiveness
Li Gu, et al.
Cell Metabolism, 2023, doi: 10.1016/j.cmet.2023.03.021
Products used:
- Adeno-Associated Virus (AAV) Packaging (AAV8)
- Vector Cloning
Abstract:Insulin inhibits gluconeogenesis and stimulates glucose conversion to glycogen and lipids. How these activities are coordinated to prevent hypoglycemia and hepatosteatosis is unclear. Fructose-1,6-bisphosphatase (FBP1) is rate controlling for gluconeogenesis. However, inborn human FBP1 deficiency does not cause hypoglycemia unless accompanied by fasting or starvation, which also trigger paradoxical hepatomegaly, hepatosteatosis, and hyperlipidemia. Hepatocyte FBP1-ablated mice exhibit identical fasting-conditional pathologies along with AKT hyperactivation, whose inhibition reversed hepatomegaly, hepatosteatosis, and hyperlipidemia but not hypoglycemia. Surprisingly, fasting-mediated AKT hyperactivation is insulin dependent. Independently of its catalytic activity, FBP1 prevents insulin hyperresponsiveness by forming a stable complex with AKT, PP2A-C, and aldolase B (ALDOB), which specifically accelerates AKT dephosphorylation. Enhanced by fasting and weakened by elevated insulin, FBP1:PP2A-C:ALDOB:AKT complex formation, which is disrupted by human FBP1 deficiency mutations or a C-terminal FBP1 truncation, prevents insulin-triggered liver pathologies and maintains lipid and glucose homeostasis. Conversely, an FBP1-derived complex disrupting peptide reverses diet-induced insulin resistance.
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SERCA2 phosphorylation at serine 663 is a key regulator of Ca2+ homeostasis in heart diseases
Fabrice Gonnot, et al.
Nature Communications, 2023, doi: 10.1038/s41467-023-39027-x
Products used:
- Adeno-Associated Virus (AAV) Packaging (AAV9)
- Vector Cloning
Abstract:Despite advances in cardioprotection, new therapeutic strategies capable of preventing ischemia-reperfusion injury of patients are still needed. Here, we discover that sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA2) phosphorylation at serine 663 is a clinical and pathophysiological event of cardiac function. Indeed, the phosphorylation level of SERCA2 at serine 663 is increased in ischemic hearts of patients and mouse. Analyses on different human cell lines indicate that preventing serine 663 phosphorylation significantly increases SERCA2 activity and protects against cell death, by counteracting cytosolic and mitochondrial Ca2+ overload. By identifying the phosphorylation level of SERCA2 at serine 663 as an essential regulator of SERCA2 activity, Ca2+ homeostasis and infarct size, these data contribute to a more comprehensive understanding of the excitation/contraction coupling of cardiomyocytes and establish the pathophysiological role and the therapeutic potential of SERCA2 modulation in acute myocardial infarction, based on the hotspot phosphorylation level of SERCA2 at serine 663 residue.
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