2.3.1 Vector for RNAi
We used pS plasmid as the shRNA-expression vector. We used
BLOCK-iTTM RNAi Designer
(http://rnaidesigner.thermofisher.com/rnaiexpress/)
to choose some better-ranked sequences located in the CDS of theLAC1 gene (Genbank: CNBG3550), which were blasted in NCBI to
identify the most specific one as the target sequence for RNAi. We
designed two different loops (5’-TTCAAGAGA and 5’-CTCGAG) linking two
reverse target sequences to compose two types of double-strands shRNA
genes, namely LAC1-RNAi-A-F/-R and LAC1-RNAi-B-F/-R (Supplementary Table
1). The designed single-strand shRNA genes were dissolved with
ddH20 (100 μM) and 2 μL of each were mixed with 5 μL 10
x PCR buffer and 41 μL ddH20 at 95 ℃ for 5 min before
cooling down at room temperature for 1 h. Two types of the
double-strands shRNA genes hybridized respectively. The hybridized
oligonucleotides contained sticky ends at 5’ end of each strand. At the
same time, the pSilencer plasmid was digested in a 20 μL system
containing pS plasmid 6μL, 10 x buffer 2μL, HindIII 2 μL, BamHI 2 μL,
ddH20 8 μL. After digested, the hybridized
oligonucleotides ligated to the long linear segments of the pS plasmid
by using T4 DNA ligase at 16 ℃ for 16 h. The pS plasmid ligated with
hybridized oligonucleotides (pS-LAC1-A and pS-LAC1-B, shown in Fig. 2A)
and the empty pS plasmid were transformed into E. coli DH5α
competent cells incubated on ampicillin-containing agar plates at 37 ℃
for 16 h. Finally, three random colonies were picked out for sequencing
by primers M13-F and CMV-F to confirm the successful construction of the
pS-LAC1 vectors (its sequencing was displayed in Supplementary Fig. 2,
sequence 1 and 2).