Syncrip facilitates porcine parvovirus viral dna replication through the alternative splicing of ns1 mrna to promote ns2 mrna formation



tải về 5.65 Mb.
Chế độ xem pdf
trang5/18
Chuyển đổi dữ liệu07.01.2023
Kích5.65 Mb.
#54118
1   2   3   4   5   6   7   8   9   ...   18
PPV.NS

Antibodies and reagents
Antibodies include monoclonal mouse anti-SYNCRIP 
antibody (ab10687; abcam, Cambridge, MA, UK), 
monoclonal mouse anti-capsid primary antibody 3C9 
(3C9-D11-H11; ATCC, Manassas, VA, USA), β-actin 
(13C000500; GenScript, Nanjing, China), Normal mouse 
IgG antibody (A7028; Beyotime, Shanghai, China), HRP-
conjugated anti-mouse IgG (RK244131; Invitrogen, 
Waltham, MA, USA). T7-Biotin Labeling Transcrip-
tion Kit (R11074.1; Ribo, Guangzhou, China), Dyna-
beads

MyOne

streptavidin C1 (65002; ThermoFisher, 
Waltham, MA, USA), Protein A PLUS-Agarose and Pro-
tein G PLUS-Agarose (SC-2002; Santa, Beijing, China).
CRISPR‑Cas9 mediated SYNCRIP knockout in PK‑15 cells
Targeting sites in the SYNCRIP gene were selected using 
the CRISPR program (Genome Engineering, Broad Insti-
tute Cambridge, MA, USA). CRISPR-Cas9 designing and 
analysis methods were described in a previous study [
30

31
]. In brief, two pairs of gRNA specifically targeting the 
porcine SYNCRIP sequence were designed using the 
optimized CRISPR design tool. Oligonucleotides were 
annealed and ligated into the Lenti-CRISPRv2 plasmid 
(Addgene, #52961) using the BsmB I sites, respectively, 
and confirmed by sequencing analysis. HEK293T cells 
were transfected with recombinant plasmids accom-
panied with psPAX2 (Addgene, #12260) and pMD2.G 


Page 4 of 15
Chen et al. Vet Res (2021) 52:73 
(Addgene, #12259) to obtain recombinant lentiviruses 
at 72 h. Then, the culture supernatant was collected 
and used to infect PK-15 cells. After 48 h, the cells were 
selected by puromycin at a concentration of 10 μg/mL. 
Positive cells were obtained after about 2 weeks and then 
subcloned into 96-well plates for single-clone growth and 
saved as cell stocks.

tải về 5.65 Mb.

Chia sẻ với bạn bè của bạn:
1   2   3   4   5   6   7   8   9   ...   18




Cơ sở dữ liệu được bảo vệ bởi bản quyền ©hocday.com 2023
được sử dụng cho việc quản lý

    Quê hương