5. Turbo DNA-free DNase treatment and removal reagents
(Ambion).
6. oligo(dT) primer and SuperScript II reverse transcriptase (Life
Technologies).
7. Gene-specific primers for qPCR of beta-interferon [21] and
β-actin [37].
2.5.6 Vaccine Efficacy
Study
1. 12 Female BALB/c Mice.
2. Phosphate-buffered saline (PBS). (see Subheading 2.3.1, item
6) (see Note 1).
3. MA-WT virus, PR8, HK68-MA20C [33].
4. 10% formalin for fixation.
5. Monoclonal antibody against the NP (clone HB65; ATCC) of
influenza A virus.
2.5.7 Protein Expression
Studies Using Western
Blotting
1. 12% SDS–polyacrylamide gels.
2. Primary antibodies.
(a) PB2 (catalog number vN19; Santa Cruz).
(b) PB1 (catalog number F5-19; BEI Resources).
(c) PA (a generous gift from Ervin Fodor, University of
Oxford), HA (catalog number ab91531; Abcam).
(d) NP (catalog number C43; Abcam).
(e) NA (catalog number ab21304; Abcam).
(f) M1 (catalog number GA2B; Santa Cruz).
(g) NS1 (catalog number 23-1; Santa Cruz).
(h) β-actin (catalog number C4; Santa Cruz).
3. IRDye-conjugated secondary antibodies (LI-COR).
4. Odyssey CLx infrared imaging system.
2.6 Transposon
Mutagenesis [16]
2.6.1 Cell Culture
1. Cell line: HEK293T, MDCK cells.
2. DMEM.
3. 5% FBS.
4. Penicillin/streptomycin (100 U/mL and 50 μg/mL,
respectively).
5. 1 mM sodium pyruvate.
2.6.2 Generation of M
Gene Segment Mutant
Plasmid Library
and Functional Profiling
1. 15-nt sequence (5
0 -NNNNNTGCGGCCGCA-3
0 ; N ¼ duplicated 5 nucleotides from target DNA).
2. (MGS kit, Finnzymes).
3. E. coli DH10B.
4. ElectroMax DH10B, Invitrogen.
Live Vaccine Attenuation
337
(Ambion).
6. oligo(dT) primer and SuperScript II reverse transcriptase (Life
Technologies).
7. Gene-specific primers for qPCR of beta-interferon [21] and
β-actin [37].
2.5.6 Vaccine Efficacy
Study
1. 12 Female BALB/c Mice.
2. Phosphate-buffered saline (PBS). (see Subheading 2.3.1, item
6) (see Note 1).
3. MA-WT virus, PR8, HK68-MA20C [33].
4. 10% formalin for fixation.
5. Monoclonal antibody against the NP (clone HB65; ATCC) of
influenza A virus.
2.5.7 Protein Expression
Studies Using Western
Blotting
1. 12% SDS–polyacrylamide gels.
2. Primary antibodies.
(a) PB2 (catalog number vN19; Santa Cruz).
(b) PB1 (catalog number F5-19; BEI Resources).
(c) PA (a generous gift from Ervin Fodor, University of
Oxford), HA (catalog number ab91531; Abcam).
(d) NP (catalog number C43; Abcam).
(e) NA (catalog number ab21304; Abcam).
(f) M1 (catalog number GA2B; Santa Cruz).
(g) NS1 (catalog number 23-1; Santa Cruz).
(h) β-actin (catalog number C4; Santa Cruz).
3. IRDye-conjugated secondary antibodies (LI-COR).
4. Odyssey CLx infrared imaging system.
2.6 Transposon
Mutagenesis [16]
2.6.1 Cell Culture
1. Cell line: HEK293T, MDCK cells.
2. DMEM.
3. 5% FBS.
4. Penicillin/streptomycin (100 U/mL and 50 μg/mL,
respectively).
5. 1 mM sodium pyruvate.
2.6.2 Generation of M
Gene Segment Mutant
Plasmid Library
and Functional Profiling
1. 15-nt sequence (5
0 -NNNNNTGCGGCCGCA-3
0 ; N ¼ duplicated 5 nucleotides from target DNA).
2. (MGS kit, Finnzymes).
3. E. coli DH10B.
4. ElectroMax DH10B, Invitrogen.
Live Vaccine Attenuation
337