Lentivirus Packaging

재조합 렌티바이러스(lentivirus)는 포유류 세포로 효율적인 유전자 전달을 위해 가장 흔히 사용되는 바이러스 벡터이다. 호스트 세포에서 외래 유전자의 transient·episomal 발현만 허용하는 plasmid DNA 벡터와 달리 lentivirus 벡터는 호스트 세포 게놈으로 외래 유전자를 통합하여 영구적으로 발현할 수 있다. Lentivirus는 in vivo 유전자 전달에도 효율적이다.

VectorBuilder에서는 우수한 품질의 lentivirus 패키징 서비스를 제공한다. 특히 당사는 벡터 제작 서비스에 사용되는 3세대 lentivirus 벡터 시스템에 대해 titer, 순도, 생존력 및 일관성 측면에서 재조합 lentivirus 생산 프로토콜을 크게 개선한 독점 기술과 시약을 개발했다. 그 결과 lentivirus 벡터 클로닝과 lentivirus 패키징에 대한 니즈에 만족하여 당사에 반복적으로 의뢰하는 고객들이 많아지고 있다. Research-grade lentivirus 외에도 임상 응용을 위한 GMP-grade lentivirus를 생산하여 제공하고 있다.

Types of lentivirus offered
  • VSV-G pseudotyped third-generation lentivirus (this is the default virus type)
  • VSV-G pseudotyped second-generation lentivirus
  • Lentivirus pseudotyped with other envelope proteins as requested, such as coronavirus spike (S) proteins
  • Bald lentivirus lacking viral envelope protein (this can be used as negative control in viral transduction)

Service Details

Price and turnaround
Scale Application Typical Titer Minimum Titer Volume Price (USD) Turnaround
Pilot Cell culture >4x10TU/ml >10TU/ml 250 ul (10x25 ul) $399 8-16 days
Medium >3x10TU/ml 1 ml (10x100 ul) $599
Large >2x10TU/ml >10TU/ml 1 ml (10x100 ul) $999
Ultra-purified medium Cell culture & in vivo >2x10TU/ml >10TU/ml 500 ul (10x50 ul) $1,299
Ultra-purified large 1 ml (10x100 ul) $1,599

Note:

1. The above table applies to VSV-G pseudotyped 2nd- and 3rd-generation lentivirus (integrase-deficient lentivirus (IDLV) included).

2. Click to view service details of lentivirus pseudotyped with coronavirus S protein.

3. TU = Transduction units (also known as infectious units).

Deliverables
For non-ultra-purified scales For ultra-purified scales
Your custom lentivirus Your custom lentivirus

Free: control virus

  • Pilot: >10TU/ml, 100 ul
  • Medium: >10TU/ml, 2x100 ul
  • Large: >10TU/ml, 2x100 ul

  Add-on purchase (optional): ultra-purified control virus

  • Ultra-purified medium: >10TU/ml, 10x50 ul, at $440
  • Ultra-purified large: >10TU/ml, 10x100 ul, at $540

Free: Polybrene (5 mg/ml, 200 ul)

Free: Polybrene (5 mg/ml, 200 ul)

Control virus

Control virus는 custom 바이러스의 생물학적 응용과 일치하도록 디자인되어, lentivirus transduction을 테스트하는 데 사용된다. 예를 들어 custom 바이러스가 유전자를 과발현하는 경우, 제공되는 control virus는 EGFP control lentivirus (Lentivirus overexpressing EGFP)이고, custom 바이러스가 유전자에 대해 shRNA를 발현하면 제공된 control virus는 scramble shRNA를 발현한다. Control virus에 대한 자세한 정보는 다음과 같다.

Vector System Control Virus Vector Name Control Virus Vector ID
Lentivirus gene expression system pLV[Exp]-EGFP:T2A:Puro-EF1A>mCherry VB010000-9298rtf
Lentivirus U6-based shRNA knockdown system pLV[shRNA]-EGFP:T2A:Puro-U6>Scramble_shRNA VB010000-0009mxc
Lentivirus miR30-based shRNA knockdown system pLV[miR30]-EGFP/Puro-EF1A>mCherry:Scramble_miR30-shRNA VB010000-9387zjj
Lentivirus CRISPR system pLV[Exp]-EGFP:T2A:Puro-EF1A>mCherry VB010000-9298rtf
Shipping and storage

Lentivirus는 HBSS buffer에 저장되어 있으며 드라이아이스로 배송된다. 수령 즉시, 장기보관을 위해서는 -80℃ (6개월 이상 안정), 1주일 이내 사용시는 -20℃에서 보관하여야 한다. Lentivirus의 유통기한은 약 1년이다. Lentivirus의 반복적인 동결-융해 사이클은 피하세요. 이는 상당한 titer 감소를 초래할 수 있기 때문이다.

Technical Information

Lentivirus production and QC

3세대 lentivirus 패키징의 경우 목적 유전자(GOI)를 운반하는 transfer plasmid는 당사의 독점적인 VSV-G를 인코딩하는 envelope plasmid와 Gag/Pol 및 Rev를 인코딩하는 packaging plasmid와 함께 HEK293T packaging cell에 co-transfection 된다. 48시간의 배양 후 상청액을 채취하고 원심분리하여 세포이물질을 제거한 후 여과한다. Lentivirus 입자는 이후 PEG로 농축한다. Ultra-purified lentivirus (in vivo grade)의 경우는, 바이러스 입자를 sucrose cushion ultracentrifugation으로 더욱 정제하고 농축한다. 당사는 p24 ELISA 방법을 사용하여 lentivirus titer를 측정한다.

VectorBuilder에서 생산되는 각 재조합 lentivirus에 대한 품질관리에는 titer 측정, 박테리아 및 fungi에 대한 무균 테스트, mycoplasma 검출 등이 있다. Transfer vector가 형광 단백질을 인코딩하는 경우, 해당 형광을 검출하기 위해 transduction 테스트를 수행한다. Transfer vector가 drug-selectable marker를 인코딩하면, transduction 테스트를 수행한 다음 해당 drug selection을 수행한다. 추가로 ultra-purified lentivirus의 경우, 일상적으로 endotoxin 수치를 체크하기 위해서 endotoxin 분석을 수행한다.

Technical documents
User Instructions Material Safety Data Sheet (MSDS) Certificate of Analysis (COA)

How to Order

Order both vector construction and virus packaging
Order virus packaging of your own vector

Customer-supplied vectors

If customer-supplied lentiviral plasmids are used in packaging, please send them to us following the Materials Submission Guidelines. Please strictly follow our guidelines to set up shipment to avoid any delay or damage of the materials. All customer-supplied materials undergo mandatory QC by VectorBuilder which may incur $100 surcharge for each item. Please note that production may not be initiated until customer-supplied materials pass QC.

FAQ

What features does lentivirus have comparing to other viral vectors?
Lentivirus MMLV Adenovirus AAV
Tropism Broad Broad Ineffective for some cells Depending on viral serotype
Can infect non-dividing cells? Yes No Yes Yes
Stable integration or transient Stable integration Stable integration Transient, episomal Transient, episomal
Maximum titer High Moderate Very High High
Promoter customization Yes No Yes Yes
Primary use Cell culture and in vivo Cell culture and in vivo In vivo In vivo
Immune response in vivo Low Low High Very low
How is viral titer determined in VectorBuilder?

We use the p24 ELISA for measuring lentivirus titer. This method employs a sandwich immunoassay to measure the levels of the HIV-1 p24 core protein in lentiviral supernatants. The lentivirus samples are first added to a microtiter plate, the wells of which are coated with an anti-HIV-1 p24 capture antibody, to bind the p24 in the lentivirus samples. This is followed by the addition of a biotinylated anti-p24 secondary antibody, which in turn binds to the p24 captured by the first antibody on the plate. A streptavidin-HRP conjugate is then added for binding the biotinylated anti-p24 antibody due to the interaction between streptavidin and biotin. A substrate solution is ultimately added to the samples which produces color upon interaction with HRP. The intensity of the colored product is proportional to the amount of p24 present in each lentivirus sample, which is measured by the use of a spectrophotometer and is then precisely quantified by comparing against a recombinant HIV-1 p24 standard curve. The p24 value is then correlated with the viral titer of the corresponding lentivirus sample.

What is VectorBuilder's guarantee for virus titer and turnaround?

Our titer guarantee applies to vectors for which the region being packaged into virus (from Δ5’ LTR to ΔU3/3’ LTR) is below the lentivirus cargo limit (9.2 kb). For sizes above this, it may still be possible to package the vector into virus, but the titer may be reduced. Additionally, we are not able to guarantee titer for the following vectors:

  • Vectors containing sequences that could adversely affect the packaging process such as toxic genes (e.g. proapoptotic genes), genes that compromise the integrity of packaging cells or virus (e.g. membrane proteins that cause cell aggregation), and sequences prone to rearrangements or secondary structures (e.g. repetitive or highly GC-rich sequences);
  • Customer-supplied plasmids containing undisclosed sequences or atypical lentiviral functional elements (e.g. LTR) that may introduce uncertainties to packaging efficiency.

Our estimated turnaround is the time from production initiation to completion. It does not include waiting time for any customer-supplied materials (e.g. template DNA or viral vectors), QC of such materials, and transit time for shipping final deliverables to the customer.