2.4.3 Virus Design,
Rescue, and Titration
1. Designed—wild-type PR8 (PR8-wt), PR8-control (PR8-ctl),
PR8-miR-93, and PR8-amiR-93NP.
2. PBS (see Subheading 2.3.1, item 6).
2.4.4 Mammalian Cell
Infection
1. Dulbecco’s phosphate-buffered saline (DPBS) supplemented
with Ca
2+ /Mg
2+ 0.3% BSA.
Tosyl phenylalanyl chloromethyl ketone (TPCK)-trypsin
(Sigma).
2.4.5 Northern Blot
Analysis
1. miRNeasy Mini Kit (Qiagen).
2. Probes U6 (5
0 -CACGAATTTGCGTGTCATCCTT-3
0 ).
3. miR-93 (5
0 -CTACCTGCACGAACAGCACTTTG-3
0 ).
4. amiR-93NP (5
0 -GAGGCTTCTTTATTCTAGG-3
0 ).
5. Highly Sensitive miRNA Northern Blot Assay Kit (Signosis).
6. Chemiluminescent HRP substrate (Takara Bio).
7. Image Quant LAS400 (GE Healthcare).
2.4.6 Western Blot
Analysis
1. 10% SDS-PAGE.
2. Nitrocellulose membrane.
3. Semidry transblot apparatus (Bio-Rad).
4. PBS with 1% Tween (PBST), 5% nonfat milk.
5. Anti-NP monoclonal antibody (Abcam).
6. Alkaline phosphatase–conjugated goat anti-mouse IgG antibody (Cell Signaling).
7. Chemiluminescent HRP substrate.
2.4.7 Virulence test
in vivo
1. Mice (6–8-week-old) purchased from Jackson Laboratory.
2. DPBS.
3. Ketamine and xylazine as anaesthetics.
2.5 Exploiting Codon
Usage Bias for Viral
Attenuation [15]
2.5.1 Development of a
Human Influenza Virus
Having Avian Influenza
Virus-like Codon Bias
Sequences
1. Influenza A/Brisbane/59/2007 (H1N1) (BR59).
2. GenScript.
Live Vaccine Attenuation
335
Rescue, and Titration
1. Designed—wild-type PR8 (PR8-wt), PR8-control (PR8-ctl),
PR8-miR-93, and PR8-amiR-93NP.
2. PBS (see Subheading 2.3.1, item 6).
2.4.4 Mammalian Cell
Infection
1. Dulbecco’s phosphate-buffered saline (DPBS) supplemented
with Ca
2+ /Mg
2+ 0.3% BSA.
Tosyl phenylalanyl chloromethyl ketone (TPCK)-trypsin
(Sigma).
2.4.5 Northern Blot
Analysis
1. miRNeasy Mini Kit (Qiagen).
2. Probes U6 (5
0 -CACGAATTTGCGTGTCATCCTT-3
0 ).
3. miR-93 (5
0 -CTACCTGCACGAACAGCACTTTG-3
0 ).
4. amiR-93NP (5
0 -GAGGCTTCTTTATTCTAGG-3
0 ).
5. Highly Sensitive miRNA Northern Blot Assay Kit (Signosis).
6. Chemiluminescent HRP substrate (Takara Bio).
7. Image Quant LAS400 (GE Healthcare).
2.4.6 Western Blot
Analysis
1. 10% SDS-PAGE.
2. Nitrocellulose membrane.
3. Semidry transblot apparatus (Bio-Rad).
4. PBS with 1% Tween (PBST), 5% nonfat milk.
5. Anti-NP monoclonal antibody (Abcam).
6. Alkaline phosphatase–conjugated goat anti-mouse IgG antibody (Cell Signaling).
7. Chemiluminescent HRP substrate.
2.4.7 Virulence test
in vivo
1. Mice (6–8-week-old) purchased from Jackson Laboratory.
2. DPBS.
3. Ketamine and xylazine as anaesthetics.
2.5 Exploiting Codon
Usage Bias for Viral
Attenuation [15]
2.5.1 Development of a
Human Influenza Virus
Having Avian Influenza
Virus-like Codon Bias
Sequences
1. Influenza A/Brisbane/59/2007 (H1N1) (BR59).
2. GenScript.
Live Vaccine Attenuation
335