2. MATERIALS AND METHODS
2.1. Plasmid construction
The plasmids expressing Cas9 and sgRNAs targeted to ovalbumin distal
promoter (px459-14 & px459-15) and that targeted to ovalbumin exon 2
(px459-6) were designed using the CRISPR design tool
(http://crispr.mit.edu/) and then generated by routine subcloning
techniques (Table 1). The donor vector for CRISPR HDR (homology-directed
repair) (pHD_4520) was generated by ligating a PCR-amplified, 556 bp
DNA ovalbumin fragment (beginning of the exon 2) as the 5′ homology arm,
and a PCR-amplified, 526 bp DNA ovalbumin fragment as the 3′ homology
arm (Table 1).
2.2.Targeted deletion of ovalbumin promoter in cultured
DF1
cells
DF1 cells were received as a generous gift from Dr. Bertrand Pain (The
Stem cell and Brain Research Institute, Lyon, France), and cultured as
recommended by the ATCC. In order to perform CRISPR excision of
ovalbumin promoter with dual sgRNAs, DF1 cells were transfected with
px459-14 and px459-15 (Table 1) using Lipofectamine 3000 (Invitrogen,
USA), as described previously (Abu-Bonsrah, Zhang, & Newgreen, 2016).
Briefly, 0.5 μg from each of px459-14 and px459-15 plasmids were diluted
with 50 μl OPTI-MEM + GlutaMax (Thermo Fisher Scientific, USA) and was
mixed with 50 μl OPTI-MEM + GlutaMax containing 1 μl Lipofectamine 3000
reagent and then incubated with 0.1–0.15 × 106 DF1
cells for 4 hours. Subsequently, the cells were cultured in 500 μl of an
antibiotic-free DMEM-F12 culture medium (Thermo Fisher Scientific) and
incubated for 24 h at 38°C in a 7.5% CO2 environment.
The transfectd DF1 cells were exposed to puromycin dihydrochloride (2.5
μg/ml; Sigma-Aldrich, USA) for 3 days. DF1 cells after antibiotic
exposure were expanded for 2 to 3 weeks. A mixed population of these
cells was subjected to genomic PCR to confirm the deletion of ovalbumin
distal promoter in a fraction of cells. After single-cell isolation and
clonal expansion, 3 clones of distal ovalbumin promoter knockout DF1
cells (DF1 +/OVA Pro ∆) were acquired that showed
correct deletion in the ovalbumin distal promoter by PCR. These 3 clones
were analyzed for the expression of ovalbumin by RT-qPCR (see below).
2.3.Confirmation of the ovalbumin distal promoter
deletion
Genomic DNA was extracted from wild-type and distal ovalbumin promoter
knockout DF1 cells (DF1 +/OVA Pro ∆) using the Genomic
DNA Extraction Kit (DENAzist Asia Co., Iran). Gene-targeting events were
detected by a single-round or nested PCR using the designed primers
(Table 2) and Taq DNA polymerase master mix RED (Ampliqon, Denmark), and
Sanger sequencing of the amplicons (Genomin Co., Iran).
2.4.Analysis of Ovalbumin expression in DF1 cells with
the deletion of distal ovalbumin promoter
Total RNA was isolated from magnum tissue (from a 35-week old laying
hen), wild-type, and distal ovalbumin promoter knockout DF1 cells (DF1+/OVA Pro ∆) using the Total RNA Isolation Kit
(DENAzist Asia, Iran). The quality and quantity of the extracted RNA
were evaluated using gel electrophoresis and a 2000 Nanodrop
spectrophotometer (Thermo Scientific, USA). One μg of total RNA was
reverse transcribed using random hexamer primers and MMLV reverse
transcriptase (Thermo Fisher Scientific, USA). To quantify the level of
transcripts for Ovalbumin and Gapdh genes, quantitative
RT-PCR reactions containing 1x SYBR Green Real-time PCR Master Mix
(Thermo Scientific, USA), 2 μl cDNA template and each primer (Table 2,
Supplementary Figure 3) at 250 nM in a 20 μl reaction volume, were
carried out in a Rotor-Gene Q real-time PCR cycler (Qiagen, USA).
Amplification steps were: 95°C for 15 min, followed by
35 cycles of 95°C for 30 s, 58°C for
30 s, and 72°C for 30 s. To derive the melting curve,
the temperature was increased in steps of 0.2°C for 5 s from 55°C to
95°C. To confirm the identity of products, PCR-amplified bands after
clean-up and reaction recovery (DENAzist Asia Co., Iran), were subjected
to Sanger sequencing (Genomin Co., Iran).
To adjust the reaction temperature, to find the best concentration of
primers, and to optimize the amplification and melting curves
(Supplementary Figure 1A), qPCR reactions were repeated. The identity of
qPCR products (Supplementary Figure 1B) was confirmed by Sanger
sequencing (Genomin Co., Iran). Complementary DNA from the magnum of the
35-week-old hen was serially diluted and subjected to qPCR to make
standard curves (Supplementary Figure 2). Each dilution was subjected to
the real-time readings in triplicate. Then, the log10 of
cDNA concentration for the Ovalbumin and Gapdh genes were
plotted against the cycle threshold (Ct) numbers to make a standard
curve (Supplementary Figure 2). To calculate the reaction efficiency,
the slope of standard curves was used in the equation of E =
(10–1/slope-1) × 100%. Both standard curves were linear in the
analyzed range with an acceptable correlation coefficient (R2)
(Supplementary Figure 2). Gene expression ratio for the Ovalbumingene over Gapdh was calculated for the magnum, wild-type DF1, and
DF1 cell with deletion of distal ovalbumin promoter using the Pfaffl
method of relative quantification (Pfaffl, 2001).
2.5.Targeted reporter knockin in DF1 cells with the
deletion of distal ovalbumin
promoter
DF1 +/OVA Pro ∆ cells were transfected with px4596 and
pHD_4520 (donor reporter vector) using Lipofectamine 3000 (Invitrogen,
USA), as described above. The cells 48 hr after transfection were
subjected to antibiotic selection with puromycin dihydrochloride (2.5
μg/ml; Sigma-Aldrich, USA). To confirm knockin of the reporter construct
(DsRed2-CMV-Puro-IRES-EGFP), genomic PCR and Sanger sequencing (Genomin
Co., Iran) were performed. Cells with the inserted reporter and deleted
ovalbumin promoter (DF1 +/OVA Pro ∆-Tg (promoterless
dsRed)) were observed and photographed by fluorescence microscopy
(Nikon Eclipse Ts2R, Japan) 2 weeks after transfection.