Background
Results
Plasmid construction
Plasmid type | Plasmid (related properties) |
ori and marker |
---|---|---|
Vector plasmid | pcDNA3.1(−) (CMV, SV40 pA) | pUC ori, ApR
|
T-vector precursor | pYA4518 (AhdI-GFP cassette-AhdI) | p15A ori, CmR
|
Reporter plasmid | pYA4392 (CPI-EGFP), pYA4924 (MPI-EGFP) | |
pYA4332 (GFP cassette) | pUCori, ApR
| |
pYA4731 (CMV-mCherry) | ||
pYA4732 (CMV-mCherry, PB2, PB1, PA, NP, HA, NA, M, NS) | p15A ori, CmR; | |
MPI-based bidirectional vector | pYA4963 (CMV-MPI), pYA4964 (CMV-GFP cassette-MPI) | pUCori, ApR
|
CPI-based 8-plasmid system | pYA4383 (PB2), pYA4384 (PB1), pYA4385 (PA), pYA4386 (NP), pYA4388 (HA), pYA4389 (NA), pYA4390 (M), pYA4391 (NS) | |
MPI-based 8-plasmid system | pYA4965 (PB1), pYA4966 (PB2), pYA4967 (PA), pYA4968 (NP), pYA4969 (HA), pYA4970 (NA), pYA4971 (M), pYA4972 (NS) | |
1-unit plasmid | pYA4973 (PB1), pYA4974 (NP), pYA4975 (NA), pYA4976 (NS); pYA4977 (PB1,GFP), pYA4978 (NP, GFP), pYA4979 (NA,GFP), pYA4980 (NS,GFP) | p15A ori, CmR
|
2-unit plasmid | pYA4981 (PB1,PB2), pYA4982 (NP,PA), pYA4983 (NA,HA), pYA4984 (NS,M), pYA4985 (PB1,PB2,GFP), pYA4986 (NP,PA,GFP), pYA4987 (NS,M,GFP) | |
4-unit plasmid | pYA4988 (NP, PA, PB1, PB2,GFP), pYA4989 (NS, M, NA, HA) | |
8-unit plasmid | pYA4990 (NP, PA, PB1, PB2, NS, M, NA, HA) | |
6-unit plasmid | pYA5000 (NP, PA, PB1, PB2, NS, M) |
Plasmid stability in E. coli
Virus generation
Cell | Plasmid | Transfection | |||||
---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | ||
BHK-21 | “1 + 2” plasmidsa
| 3 × 105
| 1 × 105
| 1 × 105
| - | - | - |
pYA4990 | 1 × 105
| 3 × 105
| 3 × 105
| 6 × 106
| 3 × 106
| 6 × 106
| |
pYA5000 | 0 | - | - | - | - | - | |
CHO | pYA4990 | 10 | 0 | 10 | - | - | - |
pYA5000 | 0 | - | - | - | - | - | |
MDCK | “1 + 2” plasmids | 0 | 0 | 0 | - | - | - |
pYA4990 | 3 × 105
| 3 × 105
| 0 | 3 × 103
| 1 × 106
| 3 × 103
| |
pYA5000 | 0 | - | - | - | - | - |
Transfection efficiency
Discussion
Conclusions
Methods
Bacterial strain and cell lines
Plasmid construction
Target/template | Primer | Sequence (5′ → 3′)a
|
---|---|---|
MTI-EGFP/pYA4392 | P1 | ttgtcgcccggagtactggtcg |
P2 | attggacctggagataggtagtagaaacaaggtag | |
MPI/mouse genomic DNA | P3 | ctaccttgtttctactacctatctccaggtccaat |
P4 | tacgtctgaggccgagggaaagc | |
SV40 pA/pcDNA3.1(−) | P5 | tacagacatgataagatacat |
P6 | cctcggcctcagacgtaaacttgtttattgcagc | |
CMV/pcDNA3.1(−) | P7 | caacttcggaggtcgaccagtactccgggcgacaagagctctgcttatatag |
P8 | ttaagatctgtacatcaatgggcgtgg | |
MPI/pYA4924 | P9 | gctgcaataaacaagtttacgtctgaggccgagg |
P10 | tggtcgacctccgaagttgggggggtgagacggatccgtctctacctatctccaggtcc | |
GFP cassette/pYA4332 | BglII-lpp | taaagatctttgttgtgtgaattaat |
BglII-5ST1T2 | ttaagatcttccattattgaagcatt | |
PB1/pYA4384 | AarI-1 PB1 | taacacctgcagtcaggtagtagaaacaaggcatt |
AarI-2 PB1 | ttacacctgcgactggggagcgaaagcaggcaaac | |
PB2/pYA4383 | AarI-1 PB2 | taacacctgcagtcaggtagtagaaacaaggtcgt |
AarI-2 PB2 | ttacacctgcgactggggagcgaaagcaggtcaat | |
PA/pYA4385 | BsmBI-1PA | taacgtctctaggtagtagaaacaaggtact |
BsmBI-2PA | ttacgtctctggggagcgaaagcaggtactg | |
NP/pYA4386 | BsmBI-1NP | taacgtctctaggtagtagaaacaagggtat |
BsmBI-2NP | ttacgtctctggggagcaaaagcagggtaga | |
HA/pYA4388 | BsmBI-1HA | taacgtctctaggtagtagaaacaagggtg |
BsmBI-2HA | ttacgtctctggggagcaaaagcaggggaa | |
NA/pYA4389 | AarI-1NA | taacacctgcagtcaggtagtagaaacaaggagtt |
AarI-2NA | ttacacctgcgactggggagcgaaagcaggagttt | |
M/pYA4390 | BsmBI-1 M | taacgtctctaggtagtagaaacaaggtagt |
BsmBI-2 M | ttacgtctctggggagcaaaagcaggtagat | |
NS/pYA4391 | BsmBI-1NS | taacgtctctaggtagtagaaacaagggtgt |
BsmBI-2NS | ttacgtctctggggagcaaaagcagggtgac | |
NP cassette /pYA4968 | PmlI-NP |
cacgtgtacatcaatgggcgtggatagcg |
SmaI-NgoMIV-NP |
cccgggatagccggcagacatgataagatacat | |
PA cassette/pYA4967 | NgoMIV-PA | taagccggcgtacatcaatgggcgtggat |
GGG-AsiSI-PA |
gggatagcgatcgcagacatgataagatacat | |
PB1 cassette/pYA4965 | AsiSI-PB1 |
gcgatcgcgtacatcaatgggcgtggat |
SmaI-NotI-PB1 |
cccgggatagcggccgcagacatgataagatacat | |
PB2 cassette/pYA4966 | NotI-PB2 | taagcggccgcgtacatcaatgggcgtggat |
GGG-BssHII-PB2 |
gggatagcgcgcagacatgataagatacat | |
NS cassette/pYA4972 | BssHII-NS |
gcgcgcgtacatcaatgggcgtggatag |
SmaI-KpnI-NS |
cccgggataggtaccagacatgataagatacat | |
M cassette/pYA4971 | KpnI-M | taaggtaccgtacatcaatgggcgtggat |
GGG-PacI-M |
gggatattaattaacagacatgataagatacat | |
NA cassette/pYA4970 | PacI-NA |
ttaattaagtacatcaatgggcgtggatag |
SmaI-SbfI-NA |
cccgggatacctgcaggcagacatgataagatacat | |
HA cassette/pYA4969 | SbfI-HA | taacctgcagggtacatcaatgggcgtggat |
GGG-HA |
gggcagacatgataagatacattgatg | |
GFP cassette/pYA4964b
| GGG-5ST1T2 |
gggtccattattgaagcatttatcaggg |
SbfI-lpp | taacctgcaggttgttgtgtgaattaatttgt | |
KpnI-lpp | taaggtaccttgttgtgtgaattaatttgt | |
NotI-lpp | taagcggccgcttgttgtgtgaattaatttgt | |
NgoMIV-lpp | taagccggcttgttgtgtgaattaatttgt | |
PacI-lpp | taattaattaattgttgtgtgaattaatttgt | |
BssHII-lpp | taagcgcgcttgttgtgtgaattaatttgt | |
AsiSI-lpp | taagcgatcgcttgttgtgtgaattaatttgt |