TABLE 1.
Primers used for generation of expression plasmids and for construction of MDV-1 mutants and rescuant viruses
| Primer | Sequencea | Fragment (plasmid generated) |
|---|---|---|
| GST-gE1 | 5′-ggattcGTAGGAGACGACGTCGCACG-3′ | GST-gE fusion protein |
| GST-gE2 | 5′-ctcgagTCAGTGGTATAAATCTAAGCG-3 | GST-gE fusion protein |
| gI-a | 5′-CAAtctagaACGGCGTGTGTGATTGCGATG-3′ | 1.5-kbp gI ORF (pcMgI) |
| gI-b | 5′-CAAgggcccCCACCTACCTATAATAGTTTC-3′ | 1.5-kbp gI ORF (pcMgI) |
| gE-a | 5′-CATAAgcatgcGAGTCAGCGTCATAATGTG-3′ | 1.1-kbp gE ORF (pcMgE) |
| gE-b | 5′-CAAgggcccATCAGTGGTATAAATCTAAGC-3′ | 1.1-kbp gE ORF (pcMgE) |
| gI-kanR-a | 5′-TCTATAGTTTATACTGGAACATCTGTTACGTTATCAACGGACCAATCTG CGATTTATTCAACAAAGCCACG-3′b | Kanamycin resistance gene for gI and gEI deletion |
| gI-kanR-b | 5′-CACATTCTTCTCTTTCCAACAATTCGACTTTCTTCTTCAGTTTTTCCATC GCCAGTGTTACAACCAATTAACC-3′b | Kanamycin resistance gene for gI deletion |
| gE-kanR-a | 5′-ATGTGTGTTTTCCAAATCCTGATAATAGTGACGACGATCAAAGTAGCTGG CGATTTATTCAACAAAGCCACG-3′b | Kanamycin resistance gene for gE deletion |
| gE-kanR-b | 5′-CTAAGCGTTTCCTAATTTTCGGCATATCATTTTTTAGCCAAGCGGTATAAG CCAGTGTTACAACCAATTAACC-3′b | Kanamycin resistance gene for gE and gEI deletion |
| gI-rev-a | 5′-TTCAAATCACCTGACGACGA-3′ | Fragment for gE rescuant |
| gE-rev-b | 5′-TAACTCTTCCAACTCCAGGG-3′ | Fragment for gI rescuant |
a
Restriction enzyme sites are given in lowercase boldface type; sequences in italics indicate additional bases which are not present in the MDV-1 sequence.
b
For primers gI-kanR-a, gI-kanR-b, gE-kanR-a, and gE-kanR-b, underlined sequences indicate sequences from pACYC177 used to amplify the kan resistance gene. Sequences printed in boldface italic type represent gI and gE sequences which allowed homologous recombination for recE/T-mediated deletion of the genes.