• 높은 역가의 바이러스 생산을 위한 최적의 조합
  - 실험 최적화를 위한 시간/시약 소모 불필요
• 편리한 Single shot 형태로, hands-on time을 최소화하고, error 방지
  - Single Shot에 선택한 transfer plasmid (ZsGreen1 발현/luciferase 발현)와 D.W.를 첨가하여 packaging cell에 처리하는 것 만으로 packaging 완료
• SARS-CoV-2 spike protein variant에 따른 제품 선택
  -> WT (full length) - Wuhan-Hu-1 (NC_045512.2)로부터 유래한 SARS-CoV-2 서열을 이용했으며, mammalian cell에서 발현될 수 있도록 최적화.
  -> D614G (full length) - 현재 유행하는 대부분의 바이러스에서 보이는 D614G mutation spike protein coding sequence 포함 (Isabel et al. 2020).
  -> B.1.351 (full length) - South Africa로부터 유래된 것으로 확인되는 B.1.351 mutation spike protein coding sequence 포함 (Johnson et al. 2020).
  -> WT (truncated) / D614G (truncated) - WT spike와 D614G, B.1.351 spike coding sequence에서 C-termini의 ER retention signal을 제거하여 바이러스가 발현하는 spike protein이 증가하고, 높은 감염력 확인 (Johnson et al. 2020)
• Neutralization assay를 위한 no-envelope control 제공
• Human ACE2 유전자를 높게 발현하는 293T cell line
  - 바이러스와 receptor간의 상호작용이나 바이러스 감염 경로 등 확인

Cell Line 취급 주의사항 (클릭)

제품설명

Streamlined, one-step workflow

Lenti-X™ SARS-CoV-2 Packaging Single Shots은 Xfect™ transfection 시약과 Lentiviral packaging plasmid가 최적의 비율로 혼합되어 동결건조 형태로 제공된다. 얻고자 하는 바이러스의 spike protein variant를 포함하는 single shot을 선택할 수 있고, 바이러스 도입 효율 확인을 위한 reporter가 삽입되어 있는 transfer vector (ZsGreen1 혹은 luciferase)와 D.W를 single shot과 섞어 packaging cell (e.g. Lenti-X™ 293T Cell Line)에 넣는 것 만으로 packaging이 완료된다. Transfection 후 48-72시간 후에 상층액에서 바이러스를 회수할 수 있다.

제품 성능 확인

그림 1. ACE2-positive cell에서의 SARS-CoV-2 pseudovirus 도입 효율 확인
Human ACE2 receptor를 안정적으로 발현하는 HEK293T cell에 SARS-CoV-2 pseudovirus를 도입한 후, ZsGreen1을 이용해 각 spike protein 별 도입 효율을 비교하였다.

(Panel A) WT spike와 D614G spike의 type 별 transduction efficiency 비교
Lenti-X SARS-CoV-2 Packaging Single Shots were used to generate lentiviral particles pseudotyped with either WT (Wuhan-Hu-1) or D614G variants of the spike protein, and encoding the fluorescent protein ZsGreen1. 100 μl of supernatant from each prep was used to transduce an HEK293T cell line stably expressing the human ACE2 receptor in the presence of 6 μg/ml polybrene in 48-well plates. Transduction efficiencies for each sample were measured by flow cytometry 6 days posttransduction.
(Panel B) B.1.351 spike와 D614G spike의 type 별 transduction efficiency 비교
Lenti-X SARS-CoV-2 Packaging Single Shots (B.1.351 or D614G spike, full length or truncated) were used to produce pseudovirus encoding the fluorescent protein ZsGreen1. Viral particles were concentrated 50-fold (B.1.351 full length) or 20-fold (B.1.351 truncated and D614G) using Lenti-X Concentrator following the standard protocol, and different volumes of each prep were used to transduce a HEK293T cell line stably expressing the human ACE2 receptor in the presence of 6 μg/ml polybrene in 48-well plates. Transduction efficiencies for each sample were measured by flow cytometry 6 days posttransduction


그림 2. ACE2-positive cell에서의 SARS-CoV-2 pseudovirus 도입 효율 확인
ACE2-positive cell과 HEK293 cell에 luciferase를 발현하는 pseudovirus를 도입한 후, 각 세포 별, spike protein 별 효율을 비교하였다.

(Panel A) WT spike와 D614G spike의 type 별 transduction efficiency 비교
Lenti-X SARS-CoV-2 Packaging Single Shots were used to generate lentiviral particles pseudotyped with either WT (Wuhan-Hu-1) or D614G variants of the spike protein, and pseudoviruses encoding firefly luciferase were used to transduce both the ACE2-positive cell line and an HEK293T cell line lacking ACE2 expression (included to provide a background signal for analysis of luciferase activity). Luciferase activity was measured 6 days posttransduction.

(Panel B) B.1.351 spikes의 type 별 transduction efficiency 비교
Lenti-X SARS-CoV-2 Packaging Single Shots (B.1.351 spike, full length or truncated) were used to produce pseudovirus encoding firefly luciferase. Viral particles were concentrated 20-fold using Lenti-X Concentrator following the standard protocol, and different volumes of each prep were used to transduce an HEK293T cell line stably expressing the human ACE2 receptor in the presence of 6 μg/ml polybrene in 48-well plates. HEK293T cells lacking ACE2 expression were transduced in parallel to determine background luminescence levels, since spike-pseudotyped viruses are incapable of infecting HEK293T cells in the absence of ACE2. Luminescence values for each sample were measured 3 days posttransduction.


그림 3. Lenti-X™ SARS-CoV-2 Packaging Single Shots로 얻은 바이러스의 functional titer 확인
(Panel A, B) Functional titers determined via analysis of ZsGreen1 reporter expression in ACE2-positive HEK293T cells transduced with SARS-CoV-2 pseudovirus (Panel A: WT spike, D614G spike / Panel B: B.1.351 spike, D614G spike)
(Panel C) Corresponding microscopy images of transduced ACE2-positive cells at 72 hours post-infection.


그림 4. SARS-CoV-2 spike protein variant에 따른 ACE2protein의 neutralizing activity 확인
(Panels A, B) ACE2 HEK293T 세포에 SARS-CoV2 pseudovirus와 ACE2-Fc 단백질을 농도별로 처리한 후, luciferase를 통해 neutralizing activity를 확인하였다.
Serial dilutions of soluble ACE2 protein fused to the Fc domain from IgG (ACE2-Fc) were applied, along with SARS-CoV-2 pseudovirus, to ACE2 HEK293T cells. Luciferase levels were measured 3 days post-infection as a readout for virus infectivity. Data are graphed as percent neutralization relative to virus-only control infection. Values are mean ±SD and experiments were performed in triplicate.

참고 문헌

제품 구성