Figure 2

Rescue and stability of the HAV constructs containing antibiotic resistance genes in the 2A-2B junction. (A) Subconfluent FRhK4 cells were transfected with in vitro T7 polymerase transcripts of pHAVvec9, pHAVvec9-Bsd, or pHAVvec9-GFP-Bsd, or pHAVvec9-Hyg or mock-transfected. Cells were and incubated for 2-weeks in selection medium containing 1 μg/ml Bsd for all transfections except for cells transfected with pHAVvec9-Hyg transcripts, which were grown in the presence of 100 μg/ml Hyg. Phase contrast micrographs were taken with a Zeiss Axiovert microscope at 200× magnification. (B) Stability of the recombinant HAV. Viral RNA was extracted and fragments were amplified by RT-PCR using HAV primers forward VP1 coding for nts 2928-2951 and reverse 2B primer coding for nts 3715-3738. RT-PCR fragments amplified from RNA extracted from HAV/7 (lane 1), HAVvec9 (lane 2), HAVvec9-Bsd passage 1 (lane 3), and HAVvec9-Bsd passage 25-times in the presence (lane 4) or absence (lane 5) of Bsd were analyzed by TAE-1%-agarose gel electrophoresis. The RT-PCR fragments from HAVvec9-Bsd and HAVvec9 are indicated by arrowheads and their sizes given in base pairs (bp). The size of the DNA molecular weight markers (lane M) is indicated in bp.