As lentiviruses pseudotyped with a Sindbis envelope M168 can target specific cells for infection via conjugated antibodies (Morizono et al., 2005), we used these modified lentiviral vectors to generate transgenic chickens. The VSV-G-pseudotyped lentivirus control used in this study has become a benchmark for assessing the efficiency of transduction by other viral envelope pseudotypes (Mochizuki et al., 1998; Reiser et al., 1996). Lentiviral packaging systems mainly include systems with three or four plasmids. Therefore, we examined which packaging system would generate M168-pseudotyped viruses with the highest transduction efficiency (Figure 1A), measured by GFP expression. The VSV-G-pseudotyped viruses produced with four plasmids transduced 36.8% of 293T cells, less than the three-plasmid system, which had a transduction efficiency of 68.7% (Figure 1B and Supplementary Figure S1). The three-plasmid system similarly showed higher transduction efficiency for the M168-pseudotyped lentiviral vectors conjugated with HLA antibodies (Figure 1B and Supplementary Figure S1). A small amount of background infection in the non-antibody group was also observed. Therefore, the GFP-expressing M168-pseudotyped viruses generated by the three-plasmid packaging system were used in subsequent experiments.
Figure Supplementary Figure S1. Microscopy was used to detect GFP expression to assess transduction efficiency of different lentiviral packaging systems
As the ratios of the three plasmids in the viral packaging system could influence the production of viral particles, we next aimed to maximize viral particle assembly in the 293T cells. The ratios of the three plasmids (GFP-expressing vector pWPXL, lentivirus packaging plasmid psPAX2, and M168 envelope protein-expressing plasmid) were adjusted. The viral amounts were normalized to the levels of HIV-1 capsid protein p24. The most viral particles were obtained using a three-plasmid ratio of 1:2:1, with a total of 10 µg of plasmids used to package the virus (Figure 1C). This ratio was therefore used for further lentiviral production.
Poor viral transgene expression was observed in the 293T cells infected by the M168-pseudotyped lentiviruses conjugated with HLA antibodies. Therefore, we adjusted the transduction conditions to increase viral infection and therefore transgene expression in 293T cells. Different concentrations of M168-pseudotyped viral particles conjugated with 1 µg of HLA antibodies were used to transduce 293T cells. Fluorescence microscopy (Supplementary Figure S2) and flow cytometry (Figure 2A) were used to compare GFP expression to assess the efficiency of gene transduction. The cells transduced with VSV-G lentiviruses showed strong GFP expression in a dose-dependent manner; 1 µg of M168-pseudotyped viruses (normalized to p24 amount) conjugated with 1 µg of anti-HLA generated maximal GFP expression. Due to the saturation of antibody and virus conjugation, no significant differences in GFP expression were found between the cells infected with 2 µg or with 1 µg of M168 virus normalized to p24 amount. Similar results were observed using flow cytometry (33.7% GFP+ and 33.6% GFP+) and RT-PCR analyses (Figure 2A, B). Subsequently, 1 µg of viral particles normalized to p24 amount, conjugated with 1 µg of antibody, was used.
Figure Supplementary Figure S2. Fluorescent microscopy was used to detect GFP expression in cells after 48 h of virus infection
To investigate whether the M168-pseudotyped lentiviral vectors were able to specifically transduce 293T cells via the specific monoclonal antibody, HLA-ABC expression in 293T and BHK cells was analyzed. Flow cytometry results showed that 98% of 293T cells expressed HLA-ABC, and <1% of BHK cells were labeled with HLA antibodies (Supplementary Figure S3). We next determined whether transduction of 293T cells could be targeted using the M168-pseudotyped lentivirus conjugated with anti-HLA. Briefly, 293T cells, BHK cells, and a mixed population of the two cell types were transduced overnight with M168 or VSV-G lentiviruses. After 2 d, the cells were analyzed by HLA-ABC staining, followed by flow cytometry. The VSV-G lentiviruses transduced both 293T and BHK cells with efficiencies of 57.6% and 58.4%, respectively (Figure 3B). In contrast, lentiviral vectors pseudotyped with M168 and conjugated with HLA antibodies specifically infected 293T cells, but not BHK cells. These transduction results were similar to those obtained by fluorescence microscopy (Figure 3A). Thus, M168-pseudotyped lentiviruses could infect target cells under mediation of specific antibodies.
To further study the targeted infection efficiency of antibody-conjugated M168 lentiviruses, we first isolated gonads from 5.5- to 6-day-old chicken embryo (E5.5–E6) kidneys and cultured PGCs as described in the Materials and Methods (Figure 4A). The newly separated PGCs displayed a round morphology with a diameter of about 20 µm and were clear with bright edges (Figure 4B). After 8 d of cultivation, the cells clustered and grew in the suspension (Figure 4C). One of the markers specifically expressed in the germ cells was the chicken vasa homolog (CVH), which is typically used to characterize PGCs (Tsunekawa et al., 2000). RT-PCR analysis showed the expression of CVH in PGCs, but not in DF-1 cells (Figure 4C). Moreover, PGCs expressed surface markers of pluripotent stem cells, such as SSEA1, SSEA3, and SSEA4, and germ cell marker DAZL. The antibodies SSEA1, SSEA3, and SSEA4 are known to specifically bind to chicken PGCs (Jung et al., 2007; Raucci et al., 2015). Immunofluorescence staining showed that the PGC membranes were positively stained by SSEA1, SSEA3, SSEA4, and DAZL antibodies (Figure 4D). Therefore, we successfully derived PGCs from chicken gonads and characterized them with germ cell and pluripotent cell markers.
Once we established the system for specific transduction of 293T cells using HLA-mediated targeted infection, we next investigated whether we could achieve specific transduction of PGCs with M168-pseudotyped lentiviruses via mediation of a specific monoclonal antibody. Flow cytometry was used to detect three cell surface markers commonly expressed in PGCs: i.e., SSEA1 (65.9%), SSEA4 (43.2%), and EMA1 (5.12%; Figure 5A). Transduction efficiency was determined by GFP expression of the infected PGCs (Figure 5B). As a control, GFP expression was detected in PGCs and BRL feeder cells transduced with VSV-G-pseudotyped lentiviruses. Although GFP expression was detected in both PGCs and BRL cells infected with VSV-G lentiviruses, only PGCs expressed GFP after infection of M168 lentiviruses conjugated with the SSEA4 antibody. Fluorescent counting was used to quantify successfully transduced PGCs. The infection rate of PGCs by SSEA4-mediated M168-pseudotyped lentiviruses was 7.5%, which was substantially higher than the 1.7% infection rate found for VSV-G lentiviruses (Supplementary Figure S4). We also found that the SSEA4-mediated M168-pseudotyped lentiviruses only transfected PGCs, not DF-1 or feeder cells (Supplementary Figure S5). As the ZZ domain was inserted into the modified Sindbis virus E2 envelope protein, the predominant antibody molecule it binds to is IgG. Therefore, the IgM antibody of SSEA1 was not effective as a means of targeted infection, although more PGCs were positive for SSEA1 than for SSEA4 (Figure 5A, B). These results indicated that M168-pseudotyped lentiviral particles conjugated with the SSEA4 antibody could be used to transduce target PGCs in vitro.
Figure Supplementary Figure S4. Calculation of positive rate of PGCs infected by virus with fluorescent counting
We injected M168 lentiviruses conjugated with SSEA4 antibodies into the subgerminal cavity beneath the blastoderm of chicken embryos using the surrogate eggshell method to target PGCs for infection. We observed the expression of GFP in developing embryos via fluorescence microscopy. Results showed that the VSV-G lentiviruses infected various chicken embryo tissues to different degrees, and the M168 lentiviruses bearing SSEA4 antibodies induced a higher level of GFP expression in the gonads. This is a possible consequence of a lower rate of nonspecific infection (Figure 6A). Six tissues (i.e., heart, liver, kidney, muscle, gizzard, and gonad) were isolated from the embryos and then analyzed using PCR. We found that the M168-pseudotyped lentiviruses conjugated with SSEA4 antibodies targeted the gonads for infection more specifically than the VSV-G-pseudotyped lentiviruses (Figure 6B). Statistical analysis showed that more than 62.5% of embryos obtained by VSV-G-pseudotyped lentivirus infection were chimeric, but only 20% were gonadal chimeras. However, the percentage of gonadal chimeras obtained by M168-pseudotyped lentivirus infection was 50.0%–66.7%, which improved upon the rate of VSV-G lentiviruses by 30.0%–46.7% (Table 1). Therefore, M168-pseudotyped lentiviruses conjugated with SSEA4 antibodies can efficiently target PGCs for transfection in vivo, resulting in an increased rate of gonadal chimerism.
Virus Experiment No. of embryos at 6 d aNo. of chimeric embryos
(% of total embryos)
bNo. of gonadal chimeric embryos
(% of chimeric embryos)
VSV-G 1 8 5 (62.5) 1 (20) 2 15 10 (66.7) 2 (20) M168 1 13 6 (46.2) 3 (50) 2 17 6 (35.3) 4 (66.7) a: Any embryonic tissue expressing green fluorescence is categorized as a chimeric embryo.
b: Any gonadal expression of green fluorescence is categorized as a gonadal chimeric embryo.
Table 1. Rates of gonadal chimeric embryos after infection with VSV-G or M168
M168-pseudotyped lentivirus production
Optimization of gene transduction using M168-pseudotyped lentiviral vectors in 293T cells
Targeted transduction of 293T cells via antibody-conjugated M168-pseudotyped lentiviral vectors
Derivation, culture, and characterization of PGCs
Targeted transduction of PGCs by M168-pseudotyped lentiviral vectors in vitro
Targeted transduction of PGCs by M168-pseudotyped lentiviral vectors in vivo
|ZR-2019-201 Supplementary Figures S1-S5.doc|