[ORIGENE] Gene Delivery Tools Webinar Series (Part 2): Adeno-Associated Virus (AAV)

Gene Delivery Tools Webinar Series (Part 2)은 AAV (Recombinant adeno-associated virus) 소개 및 적용에 대한 웨비나입니다. 아데노바이러스는 분열세포와 비분열세포 모두에서 유전자 전달과 발현이 가능하다는 장정을 가지고 있습니다. 바이러스 벡터를 이용한 Gene delivery는 target gene의 전달 효율을 높일 수 있기 때문에 특정 세포주에 관심 있는 유전체를 전달하는 연구용 도구로 많이 사용하고 있습니다.

Part 2: AAV (Recombinant adeno-associated virus)

  1. Background on viral gene delivery
  2. AAV 101 (What, How, and Why)
  3. Safety
  4. How to produce & use AAV
  5. OriGene solutions & services

Virus

Expression

Genome

Packaging Capacity

Virus Size (nm)

Cells Infected

Target Cell Genome Integration

Immune Response

Lentivirus

Stable

RNA

< 9kb

80-130

Dividing/Non-dividing

Yes

Low

AAV

Transient or Stable*

ssDNA (linear)

~4.7kb

18-26

Dividing/Non-dividing

No*

Very Low

Adenovirus

Transient

dsDNA (linear)

>8kb

105

Dividing/Non-dividing

No

High

Y-Retrovirus

Stable

RNA

<8kb

80-130

Dividing

Yes

Moderate

*Recombinant AAV has a low frequency of target cell genome integration

ORIGENE AAV Products and Services:

Q&A :

There are several key distinctions between them, including:
1) Packaging capacity
2) Onset and duration of gene expression
3) Immune response
Adenoviruses have a capacity of ~8.5 kilobases, and onset of expression can occur as early as 16-24 hours after infection. The high immune response from the target cells is the main limitation of adenoviral systems. Despite this, they are still widely used in research, due to their highly efficient transduction of most tissues.
AAVs have a packaging capacity of ~4.5 kilobases and have a much later onset of expression (2-7 days for in vitro and 3-21 days for in vivo), however, this delivery system triggers very low levels of immune response.

You may not have produced a high enough titer. This could be caused by the health of the HEK293/HEK293T cells. HEK293T cells usually lose packaging efficiency after many passages. Cells should not be used after culturing for 1-2 months. Cells are also happier when seeded the day before transfection, which results in higher transfection efficiency.
You may also want to consider revisiting the transfection step and ensuring that you avoid contamination, have an effective transfection reagent, and lastly, consider the size of your insert

To determine the MOI needed when transducing a cell line for the first time, we recommend transducing the target cells with the same serotype eGFP reporter AAV particles at a series of MOIs, such as MOIs of 100, 1,000, and 10,000, etc…

Example: 3 x 105 HT1080 cells were prepared for transduction by AAV particles (titer: 1 x 1011 GC/ml) one day after plating. For an MOI = 1,000, 3μl AAV particles are needed. For an MOI = 10,000, 30μl AAV particles are needed.

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