5. HEK293T cells, MDCK cells.
6. TRIzol reagent (Invitrogen).
7. iScript cDNA Synthesis kit (Bio-Rad).
8. Primer Sequences.
(a) 5
0 -AGCAAAAGCAGGTAGATATT-3
0 .
(b) 5
0 -GGGGCCAAAGAAATAGCACT-3
0 .
(c) 5
0 -TCCTAGCTCCAGTGCTGGTC-3
0 .
(d) Vic-labeled
insertion-specific
mini-primer
(50 -TGCGGCCGCA-3
0 ).
9. KOD Hot-Start polymerase (Novagen).
10. Liz-500 size standard (Applied Biosystems).
11. 96-capillary genotyper (3730xl DNA Analyzer, Applied
Biosystems).
12. ABI Software.
13. PCR.
14. Ultracentrifuge.
2.6.3 Virus Strains
1. Eight plasmids harboring the cDNA of A/WSN/33
12 (a gift
from Dr. Yuying Liang at Emory University).
2. HEK293T with TranwsIT LT-1 (Panvera).
3. Influenza virus A/Victoria/3/75 (seasonal A/H3N2 virus).
4. A/Wisconsin/65/05 (seasonal A/H3N2 virus).
5. A/Hongkong/68 (seasonal A/H3N1 virus) (gifts from
Dr. Ioanna Skountzou at Emory University).
6. Influenza virus A/Cambodia/P0322095/05.
2.6.4 Virus Titration
1. Dilution buffer: PBS (see Subheading 2.6.1, item 6) (see Note
1), 10% BSA, CaCl 2 , 1% DEAE-dextran, and MgCl 2 .
2. 6-well plates.
3. Growth medium: 1% low-melting agarose, TPCK-treated trypsin (0.7 μg/mL).
4. Plaque Staining: 1% crystal violet, 20% ethanol, PBS (see Subheading 2.6.1, item 6) (see Note 1).
5. DMEM.
6. Gaussia luciferase (gLuc) reporter system (see Subheading 3.6,
step 4).
7. Renilla luciferase substrate (Promega).
2.6.5 Animals
1. Adult Mice: Female C57BL/6 mice (6–8 weeks old) (purchased from the Jackson Laboratory).
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Dipasree Hajra et al.
6. TRIzol reagent (Invitrogen).
7. iScript cDNA Synthesis kit (Bio-Rad).
8. Primer Sequences.
(a) 5
0 -AGCAAAAGCAGGTAGATATT-3
0 .
(b) 5
0 -GGGGCCAAAGAAATAGCACT-3
0 .
(c) 5
0 -TCCTAGCTCCAGTGCTGGTC-3
0 .
(d) Vic-labeled
insertion-specific
mini-primer
(50 -TGCGGCCGCA-3
0 ).
9. KOD Hot-Start polymerase (Novagen).
10. Liz-500 size standard (Applied Biosystems).
11. 96-capillary genotyper (3730xl DNA Analyzer, Applied
Biosystems).
12. ABI Software.
13. PCR.
14. Ultracentrifuge.
2.6.3 Virus Strains
1. Eight plasmids harboring the cDNA of A/WSN/33
12 (a gift
from Dr. Yuying Liang at Emory University).
2. HEK293T with TranwsIT LT-1 (Panvera).
3. Influenza virus A/Victoria/3/75 (seasonal A/H3N2 virus).
4. A/Wisconsin/65/05 (seasonal A/H3N2 virus).
5. A/Hongkong/68 (seasonal A/H3N1 virus) (gifts from
Dr. Ioanna Skountzou at Emory University).
6. Influenza virus A/Cambodia/P0322095/05.
2.6.4 Virus Titration
1. Dilution buffer: PBS (see Subheading 2.6.1, item 6) (see Note
1), 10% BSA, CaCl 2 , 1% DEAE-dextran, and MgCl 2 .
2. 6-well plates.
3. Growth medium: 1% low-melting agarose, TPCK-treated trypsin (0.7 μg/mL).
4. Plaque Staining: 1% crystal violet, 20% ethanol, PBS (see Subheading 2.6.1, item 6) (see Note 1).
5. DMEM.
6. Gaussia luciferase (gLuc) reporter system (see Subheading 3.6,
step 4).
7. Renilla luciferase substrate (Promega).
2.6.5 Animals
1. Adult Mice: Female C57BL/6 mice (6–8 weeks old) (purchased from the Jackson Laboratory).
338
Dipasree Hajra et al.