Team:Jiangnan/Demonstrate

<

1.IRF7

Though our computational modeling and network construction using public datasets retrieved from GEO（GSE76967，GSE32186，GSE40644，GSE8807，GSE8923，GSE3568） , we identified IRF7 as our candidate responsible for the high titration feature of cells. We increased virus titer over 2 folds by suppressing IRF7 expression in MDBK cells. In addition, by exposing IRF7-silenced MDBK cells to cold atmospheric plasma, we further increased virus titer to 2.5 fold. Taken, together, through genetic modification and physical plasma treatment, we could increase virus titer up to 5 folds.

Figure 1A. IRF7 gene expression with different SiRNA concentration.

Figure 1B.relative amount of viral DNA with different SiRNA concentration.

C: control NC: negative control
SiRF7-10nmol: SiRNA final concentration 10nmol/ml
SiRF7-50nmol: SiRNA final concnetration 50nmol/ml

Figure 1A showed that IRF7 was effectively knocked down, and Figure 1B showed that Bovine rhinotracheitis virus (IBRV) replication in the experimental group was over 2 folds than that in the negative control group after suppressing IRF7 expression (when siRNA concentration was 50nmol/ml).

2.Plasma

Figure 2 relative amount of viral DNA with different plasma treatment time.

After incubating IRF7 silenced cells with the DMEM medium for 1 hour, IBRV replication in MDBK was further increased 2.5 folds.

1.Transscriptome sequencing and analysis

Figure 1 MA map. The X-axis was the mean value of all sample expression used for comparison after standardization, and the Y-axis was log2 transformed fold change. The significant p-value&lt0.05 was marked in red.

Genes differentially expressed between adherent and suspended cells were mapped to KEGG pathways.

Figure 2 KEGG pathway enrichment map.

Red blocks indicate up-regulated genes, green blocks mean down-regulated genes, and the yellow one means both up-regulated and down-regulated gene.

Figure 3 Venn diagram showing the number of genes in each analytical cohort.

Figure 4 Network showing interactions among candidate genes responsible for cell suspension feature. The 18 candidate genes were shown in the inner circle and other genes relevant with these candidates were retrieved by GeneMANIA and shown in the outer circle.

In order to find the key gene responsible for cell suspension, we performed transcriptome sequencing and analysis on adherent and suspended cells from the same line and origin, with the aim of identifying genes differentially expressed between adherent and suspended cells.

2. In vitro validation of PABPC1 expression in BHK-21 adherent and suspended cells

Figure 5.Gene expression of PABPC1 in BHK-21 adherent and suspended cells.

We extracted RNA from BHK-21 adherent cells and suspension cells, and examined PABPC1 expressions in these cells by qPCR.PABPC1 level in BHK-21 suspended cells is 0.4 times of that in BHK-21 adherent cells.

3. siRNA transfection

Figure 6.Efficacy of PABPC1 siRNA at different concentrations.

Figure 7.Growth curve of BHK-21 cells with reduced PABPC1 expression.

We used siRNA to inhibit PABPC1 expression in BHK-21 adherent cells and tested the efficacy of siRNAs at different concentrations. As shown from Figure 6, siRNA at the concentration of 50umol/L could effectively reduce PABPC1 to around 0.25 fold, which was used for the following experiments.

BHK-21 adherent and suspended cells were used as the negative and positive control, respectively. After one day's adaptation, suspended and PABPC1 silenced cells entered the logarithmic growth stage. The highest cell density occurs at the 4th day which is the same between adherent and PABPC1 silenced cells and about 4x10⁶ cells/ml.

We successfully transfected the constructed biobricks into MDBK cells, the target receptors were functionally expressed on the surface of MDBK cells as validated by canine distemper virus (CDV) infection.

1. Nucleic acid gel electrphoresis

Figure 1. Construction of the Nectin 4 biobrick.
Identification of recombinant pcDNA-DogN4-FLAG.

M: DL10000 DNA Marker; 1: pcDNA-DogN4-FLAG (digested with EcoRI and XboI)

Figure 2. Construction of the TfR biobrick.
Identification of recombinant PLVX-TfR.

M: DL10000 DNA Marker; 1: PLVX-TfR (digested with EcoRI and XboI)

2. Transfection efficiency detection using inverted fluorescence microscopy after 2 days of transfection

To find the most efficient way to transfect MDBK cells, we explored four transfection methods.

The results are shown in Figure 3. For the transfection efficiency, both the lentivirus transfection method and the electroporation transfection method can meet the needs.

Figure 3. Transfection result in MDBK cells by different methods(x 100)

Figure 4. Transfection result in MDBK cells by different methods

3. Nectin 4 expression detection at the transcriptional level in steady transplanted cell strains using RT-PCR

As shown in Fig 5, Nectin 4 was expressed at the mRNA transcription level in MDBK-N4 cells.

Figure 5. The expression of NECTIN4 mRNA was dectected in MDBK-N4 cells by using RT-PCR

M:DL500 DNA Marker;1-2: MDBK cells and MDBK-N4 cells for NECTIN4;3-4: MDBK cells and MDBK-N4 cells for GAPDH

4. Nectin 4 expression detection at the translational level in steady transplanted cell strains using Western blot

Western blot results are shown in Fig 6. Compared with control MDBK cells, MDBK-N4 cells (10th generation) showed a clear band at 56 kDa, indicating that MDBK-N4 cells can stably express Nectin 4 protein.

Figure 6. Nectin 4 expression detected in MDBK-N4 cells by Western blot.

5. Nectin 4 and viral coat protein expression detection by Indirect Immunofluorescence

Immunofluorescence results are shown in Fig 7. The results indicate that MDBK-N4 cells can stably express Nectin4 and transport proteins to cell membrane.

Figure 7. Nectin4 expression detected in MDBK-N4 cells by immunofluorescence (×40).

A: MDBK cells; B: MDBK-N4 cells

Figure 8. Nectin4 expression detected in MDBK-N4 cells by immunofluorescence (×100).

A: MDBK cells; B: MDBK-N4 cells

The immunofluorescence results are shown in Fig 9. It was demonstrated that Nectin4 protein expressed by MDBK-N4 cells functioned as a virus receptor, CDV replicated and proliferated in cells to generate progeny virus, and infected surrounding cells, resulting in a large amount of cell apoptosis.

Figure 9. Envelope protein of CDV detected in MDBK-N4 cells by fluorescent microscope (×40).

A: MDBK cells; B: MDBK-N4 cells