In the present study, we studied the feasibility of deleting essential genes in insect cells by using bacmid and purifying recombinant bacmid in Escherichia coli DH10B cells. To disrupt the orf4 (open reading frame 4) gene of BmNPV [Bm (Bombyx mori) nuclear polyhedrosis virus], a transfer vector was constructed and co-transfected with BmNPV bacmid into Bm cells. Three passages of viruses were carried out in Bm cells, followed by one round of purification. Subsequently, bacmid DNA was extracted and transformed into competent DH10B cells. A colony harbouring only orf4-disrupted bacmid DNA was identified by PCR. A mixture of recombinant (white colonies) and non-recombinant (blue colonies) bacmids were also transformed into DH10B cells. PCR with M13 primers showed that the recombinant and non-recombinant bacmids were separated after transformation. The result confirmed that purification of recombinant viruses could be carried out simply by transformation and indicated that this method could be used to delete essential genes. Orf4-disrupted bacmid DNA was extracted and transfected into Bm cells. Viable viruses were produced, showing that orf4 was not an essential gene.
Homologous recombination in insect cells has long been used to delete baculovirus genes. However, recombinant baculoviruses cannot be isolated containing a knockout in essential genes or even some non-essential genes, such as the lef-6 (late expression factor 6) genes of AcNPV (Autographa californica nuclear polyhedrosis virus) and BmNPV [Bm (Bombyx mori) nuclear polyhedrosis virus] by using wild-type baculoviruses [1,2]. To date, essential genes have been deleted either by recombination in Escherichia coli through the use of bacmids or by recombination in insect cells through the use of stably transfected insect cell lines that constitutively expresses the gene to be deleted [3–6]. Bacmids contain a selectable kanamycin resistance marker and a mini-F replicon, which allows autonomous replication and stable segregation of bacmids at low-copy number . Therefore we explored the feasibility of deleting essential genes in insect cells and purifying a recombinant virus in E. coli DH10B cells by making use of the characteristics of bacmids. BmNPV orf (open reading frame) 4 is a gene whose function is completely unknown. It is 1020 bp in size and contains an early promoter motif (a TATA box, followed by a CAGT motif 20–25 bp downstream) in its promoter region . Furthermore, its expression level is high in the early stages of infection and decreases in the later stages . Accordingly, orf4 is an early gene which may play an important role in the early stage of virus infection. Using a BmNPV bacmid, which is infectious to Bm cells and silkworms , a colony containing only the orf4-disrupted bacmid was identified when bacmid DNA extracted from Bm cells was transformed into DH10B cells.
MATERIALS AND METHODS
The E. coli DH10Bac/BmNPV cell line was provided by Professor E.Y. Park (Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka, Japan) and Professor K. Maenaka (Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan) . The pBluescriptK-ie1p-EGFP plasmid containing a ie1p (immediate early 1 promoter)–EGFP (enhanced green fluorescent protein) cassette expressing EGFP under the control of the ie1p was a gift from Professor X.P. Wu (Shanghai Institute of Biochemistry, Chinese Academy of Sciences, Shanghai 200031, China) and recombinant BmNPV bacmid expressing a 500 bp fragment (orf76 of BmNPV) at the polyhedrin locus was constructed using the pFastbacHtb vector. FuGENE™ 6 transfection reagent was from Roche. Grace's insect cell culture medium (GIBCO) was purchased from Invitrogen. The B. mori cell line BmN (originated from ovary) was preserved in our laboratory and cultured at 27°C in Grace's insect cell culture medium.
Generation of recombinant viruses containing disrupted
Orf4 is located at between 3250 and 4270 bp in the BmNPV genome, with a transcriptional orientation opposite to that of the polyhedrin gene. The genomic organization of the orf4 region is shown in Figure 1(A) . The transfer vector pUCRight-armLeft-armIE1PEGFP was generated by inserting the ie1p–EGFP cassette and orf4 flanking sequences into the MCS (multicloning site) of pUC18. First, the 1854 bp ‘Right-arm’ section was PCR amplified using the primers Right-arm forward (5′-CGCTCTAGAGTGATACGACTCGCCATAC-3′), incorporating an XbaI site (underlined) and Right-arm reverse (5′-TAATGCATGCGAACACGATTTAATGCTGA-3′), incorporating an SphI site (underlined). The amplified fragment was digested with XbaI and SphI and then cloned into the XbaI and SphI sites of pUC18 to generate pUCRight-arm. Secondly, the 2079 bp ‘Left-arm’ section was PCR amplified using the primers Left-arm forward (5′-GTGGAATTCTCGTTTCTAATAGCTTCCA-3′), incorporating a EcoRI site (underlined) and Left-arm reverse (5′-GCAGGTACCAATTTTGTGTGATAACTTATG-3′), incorporating a KpnI site (underlined). The amplified fragment was digested with EcoRI and KpnI and then cloned into the EcoRI and KpnI sites of pUCRight-arm to generate pUCRight-armLeft-arm. Finally, the 1340 bp ie1p–EGFP cassette was excized from pBluescript-ie1p-EGFP by digestion of the plasmid with XbaI and KpnI, and the fragment was then gel purified and ligated into pUCRight-armLeft-arm (which had been digested previously with XbaI and KpnI) to generate the final transfer vector pUCRight-armLeft-armIE1PEGFP.
Genomic organizations of the
orf4 region of non-recombinant and recombinant bacmids
In the constructed transfer vector, a total of 436 bp in the central portion of orf4 was replaced with the ie1p–EGFP cassette, with the first 84 bp and the last 503 bp of orf4 retained. The orf4 flanking sequences, along with the retained partial orf4 coding regions, were used for homologous recombination. The transfer vector pUCRight-armLeft-armIE1PEGFP was co-transfected with BmNPV bacmid DNA into Bm cells by using Lipofectin® (Invitrogen). The resulting mixture of non-recombinant viruses and recombinant viruses containing the disrupted orf4 gene (Figure 1B) were purified once under a fluorescent microscope.
Identification of colonies harbouring only the
Bacmid DNA was extracted from Bm cells, and approx. 1 μg of bacmid DNA was transformed into DH10B competent cells. Competent cells were plated on to LB (Luria–Bertani) agar containing 50 μg/ml kanamycin, 40 μg/ml IPTG (isopropyl β-D-thiogalactoside) and 100 μg/ml X-Gal (5-bromo-4-chloroindol-3-yl β-D-galactopyranoside). PCR with the orf4 forward and reverse primers was used to screen for blue colonies harbouring only the orf4-disrupted bacmid. The primers for orf4 were as follows: forward, 5′-ATAGGATCCATGTCTCTCGCTGCAAAGT-3′; and reverse, 5′-GCGGGTACCTTGTAAATGTTTATTATTTAAAA-3′, with the BamHI and KpnI sites underlined respectively. PCR was performed under the following conditions: 1 cycle at 94°C for 5 min; 39 cycles at 94°C for 30 s, 50°C for 30 s and 72°C for 2 min 10 s; and 1 cycle at 72°C for 10 min.
Generation of recombinant viruses containing disrupted
To disrupt orf4, the transfer vector pUCRight-armLeft-armIE1PEGFP was constructed. When the transfer vector was digested with EcoRI and KpnI, the 2079 bp left-arm fragment was generated (Figure 2, lane 2). When the transfer vector was digested with KpnI and XbaI, the 1340 bp ie1p–EGFP cassette fragment was generated (Figure 2, lane 3). When the transfer vector was digested with XbaI and SphI, two fragments were generated, one being the 1854 bp right-arm section and the other being the 1574 bp fragment containing ie1p–EGFP cassette plus a part of orf4 (Figure 2, lane 4) because there is a SphI site in the last 503 bp of orf4 (Figure 1B).
Identification of the transfer vector pUCRight-armLeft-armIE1PEGFP
Homologous recombination in insect cells produces a low proportion of recombinant baculoviruses in the progeny population (usually 1–2% or less) . Moreover, most of the recombinants are single crossovers in which the entire transfer plasmid is integrated into the viral genome . Accordingly, three passages of viruses were carried out in Bm cells, followed by one round of purification under the fluorescent microscope. PCR was then performed with primers for orf4 to confirm the insertion of the ie1p–EGFP cassette into the orf4 locus and to estimate the proportion of recombinant viruses in the progeny population. The PCR product of non-recombinant viruses was 1020 bp in size, and that of recombinant viruses was approx. 1900 bp (Figure 3A, lane 2).
PCR identification of bacmid
Identification of colonies harbouring only the
Bacmid DNA was extracted from Bm cells infected with the viruses which were purified once, and transformed into competent DH10B cells, which were plated on to LB agar plates containing kanamycin. Because the bacmid carries the kanamycin resistance marker, colonies on the plate must contain the bacmid. Bacmid DNA was extracted from the colonies, and then PCR using the primers for orf4 was carried out to identify colonies harbouring the orf4-disrupted bacmid. PCR was carried out on thirteen colonies, with one colony harbouring only the orf4-disrupted bacmid and the other twelve colonies harbouring only the non-recombinant bacmid (Figure 3A, lanes 3 and 4).
Confirmation of the ability of the novel method to purify the recombinant bacmid
To confirm the ability of the novel method to purify the recombinant bacmid, a mixture of recombinant bacmid expressing a 500 bp fragment (white colonies) at the polyhedrin locus and non-recombinant bacmid expressing α-LacZ (blue colonies) were transformed into DH10B cells. PCR with M13 primers was carried out on two white colonies and two blue colonies. The result showed that the PCR product amplified from white colonies was approx. 3000 bp in size (Figure 3B, lanes 3 and 4) and that amplified from blue colonies was 300 bp (Figure 3B, lanes 5 and 6), confirming the effectiveness of the novel purification method.
Viral replication in Bm cells
To determine whether orf4 was essential for viral replication, Bm cells were transfected with the orf4-disrupted bacmid and a transfection-infection assay was performed. After transfection, Bm cells were examined for evidence of virus infection and propagation. At 5 days post-transfection, the tissue-culture supernatant was removed from transfected cells and added to a second group of freshly plated Bm cells; those cells were then incubated for 3 days to detect the presence of infectious virus generated from cells transfected with the orf4-disrupted bacmid. At each step, cells were examined for visible signs of cytopathic effects and observed by fluorescence microscopy to detect the expression of EGFP from the ie1pr. Cytopathic effects and EGFP fluorescence were observed in Bm cells transfected with the orf4-disrupted bacmid and also from cells incubated with the tissue-culture supernatant from the transfected Bm cells (Figure 4). Thus transfection-infection experiments indicated that orf4 was not essential for viral replication in Bm cells.
In order to eliminate single crossovers in recombination, serial passages of virus in insect cells should be carried out, as was performed in the present study. However, single crossovers can also be eliminated by constructing a parental vector in which a LacZ gene under the control of a T7 promoter is inserted outside of the homologous sequences. Colonies harbouring single crossovers will become blue when cultured on X-Gal-containing plates, thus distinguishing them from those harbouring double crossovers. In the present study, we used EGFP under the control of the ie1p as our selection marker, so we first purified the recombinant virus by fluorescent microscopy analysis and then used PCR to screen colonies harbouring recombinant bacmid. However, to accelerate the purification process, an antibiotic selection marker under the control of a prokaryotic promoter can be inserted inside the homologous sequences to eliminate colonies harbouring non-recombinant bacmid. The fact that a low proportion of recombinant virus in the progeny population could form colonies harbouring only recombinant bacmid when bacmid DNA was transformed into DH10B cells suggests that this method can be used to delete essential genes in insect cells, since small amounts of recombinant viruses can be rescued by the presence of non-recombinant viruses. Essential genes may be related to viral DNA replication, such as Me53 , or the formation of budded virus, such as GP64 . The ie1p is an immediately early promoter whose activity depends on no other viral factors . Therefore the ie1p is very useful in deleting essential genes. In addition, flow cytometry can be used to isolate EGFP-expressing cells when recombinant viruses carry EGFP as a selection marker.
Using the BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi), we found that orf4 and orf11 of AcMNPV [A. californica MNPV (multicapsid nucleopolyhedrovirus)] share 96% identity at the nucleotide level, and have homologues in three other baculoviruses, including Plutella xylostella MNPV, Rachiplusia ou MNPV and Maruca vitrata MNPV. The absence of orf4 homologues in other baculoviruses suggests that the role that orf4 plays may not be essential or can be provided by the hosts of these baculoviruses. The transfection-infection experiment showed that orf4 was not an essential gene in cultured cells. With its peak expression level reached at the early stage of infection, orf4 may have a function in facilitating the onset of the virus infection cycle.
This work was supported by the 973 National Basic Research Program of China [grant number 2005CB121005]; the National Natural Science Foundation of Jiangsu Education Committee [grant number 06KJD180043]; the Six-Field Top programs of Jiangsu Province; and the Innovation Foundation for Graduate Students of Jiangsu Province.