Overview

This result describes the results of the experiments performed during the project: 1. Single-round infectious particle (SRIP) production, 2. SRIP detecting cell production, and 3. SRIP infection. First, we will present the main results of these three experiments.

1. SRIP production

The plasmids we received from Dr. Suzuki at the National Institute of Infectious Diseases (NIID) was amplified in E. coli, and we were able to generate the plasmid for transfection. We will confirm SRIP production by transfection into HEK293T and C6/36 cells.

2. SRIP detecting cell production

We have confirmed that both Vero cells and C6/36 cells emit EGFP fluorescence in SRIP detecting cells. Therefore, we will perform experiments to confirm the split-Cre and Cre-LoxP systems, and aim to confirm the expression of mCherry.

3. SRIP infection

After SRIP production and SRIP detecting cell production are completed as described in 1. and 2., we will infect SRIP detecting cells with SRIP and confirm that mCherry is expressed. Furthermore, we will use neutralizing antibodies against DENV to see how SRIP infection is actually prevented.

Result

The following is a detailed description of the results presented above in the order of 1. SRIP production, 2. SRIP detecting cell production, and 3. SRIP infection.

1. SRIP production

In this section, we describe the results of our experiments to generate cells for SRIP production. This section is divided into four parts: (1) culture of HEK293T cells and C6/36 cells, (2) amplification of the transferred plasmid, (3) construction of the plasmid to be modified, and (4) transfection of the cells.

(1) Culture of HEK293T cells and C6/36 cells We first had to learn how to culture cells. We started with Vero cell culture, followed by HEK293T cell and C6/36 cell culture techniques.

Culture of HEK293T cells
The culture of the cells basically followed the Culture cells protocol (protocol). Fig. shows the cells before cryopreservation. Fig.1: Photograph of HEK293T cells P10+3 taken with a phase contrast microscope

Culture of C6/36 cells
We basically followed the Culture cells protocol for these cells as well. (protocol) Since C6/36 cells were insect cells, unlike mammalian cells that had been cultured previously, and the faculty members and graduate students who were teaching the experiment had never cultured these cells before, it was expected that it would be difficult to learn the culture technique. When we actually observed the cells, we were amazed at their size, shape, and adhesiveness. Later, we were able to obtain enough stock to proceed with the project, and Fig. shows the cells that had become sufficiently confluent.
Fig.2 : Phase contrast microscopy of C6/36 cell Pn+9

(2) Amplification of the transferred plasmid To produce SRIP, we contacted Dr. Suzuki of the National Institute of Infectious Diseases (NIID), who also helped us with the 2018 project. The plasmids we received from Dr. Suzuki is as follows.

The three plasmids we received from Dr. Suzuki are as follows.

pCMV-YF-nluc-rep (Fig.3)
This plasmid has a sequence in which the structural protein gene is deleted from the yellow fever virus genome and Nano-Luciferase is inserted as a reporter. Fig.3

pCAG-YF-C (Fig.4)
Plasmid encoding the yellow fever virus Capsid
Fig.4

pCAG D1-YG1-prME (Fig.5)
Plasmid encoding prME of DENV1
Fig.5

In order to secure these stocks, we transfected the plasmids we received into E. coli DH5α Competemt Cells and cultured them. We then extracted the plasmids from the mass-cultured E. coli.

(3) Construction of the plasmids to be modified (SRIP producting construction). We extracted the Nano-Luciferase from the pCMV-YF-nluc-rep provided by Dr. Suzuki for use in our project, and modified the C-terminal region of the Cre recombinase enzyme (hereafter referred to as "C-Cre") into a Split plasmid. C-terminal region of Cre recombinase enzyme (hereafter referred to as "C-Cre"). Fig.6 shows the electrophoresis of pCMV-YF-nluc-rep picked up and purified from three E. coli colonies and treated with SnaBI.
Fig. 6: Plasmid pCMV-YF-nluc-rep i (i= 1, 2, 3) purified from three colonies and treated with SnaBI pCMV-YF-nluc-rep i R. Markers were λ/Hind III. After treatment a band was to be seen around 11.6 kbp.

We then followed the instructions for restriction enzyme treatment with Xma I, extracted the gel, CIP'd it, and ligated it with restriction enzyme-treated synthetic DNA containing C-Cre (described below), transformed it into E. coli, and amplified it.

Synthetic DNA containing C-Cre was synthesized by Twist and treated with the restriction enzyme SnaB I. This was followed by restriction enzyme treatment with Xma I and ligation as described above.

Assuming that the constructs have been constructed in this manner, the remainder of the flow is outlined below.

(4) Transfection of cells

Plasmids prepared in this way are linearized using a restriction enzyme with only one restriction enzyme site for transfection into HEK293T cells or C6/36 cells. The reason is that the transfected gene is passed on from one generation of cells to the next. Once the cells have been successfully transfected, SRIP is obtained by harvesting the culture medium of the cells.

2. SRIP detecting cell production

In this section, we describe the experiments we performed to construct cells to detect Single-round infectious particle (SRIP) infection. The following steps are described in this section: 1) culture of Vero and C6/36 cells, 2) construction of plasmids for transfection into the cells, and 3) transfection into the cells.

(1)Culture of Vero and C6/36 cells

As mentioned earlier, we first learned how to culture Vero cells, and then began to culture HEK293T cells and C6/36 cells.

Culture of Vero cells
We followed the culture protocol of Culture cells (protocol). Fig.7 shows a picture of Vero cells before cryopreservation.
Fig.7 : Photograph of Vero cell P7 taken with a phase contrast microscope.

Culture of C6/36 cells
The culture of C6/36 cells is shown in the section of SRIP production.

(2) Plasmid Construction

The main flow of the construction is shown in SRIP detecting cell construction. This was done by inserting two types of synthetic DNA into pIRES2•EGFP to construct the gene flow of LoxP_NLS_N-Cre_IRES_EGFP_LoxP_mCherry, which is the basis of the SRIP detecting cell. Finally, by inserting LoxP_NLS_N-Cre_IRES_EGFP_LoxP_mCherry into pCAGGS as insert, we can place this sequence under the CAG promoter.

Fig.8 pCAGGS N-Cre Fig.8 shows the insert (LoxP_NLS_N-Cre_IRES_EGFP_LoxP_mCherry) and the backbone plasmid (pCAGGS) treated with two different restriction enzymes, EcoR I and Bgl II. The following figure shows a comparison of the ligated and amplified products in E. coli. However, the codon-optimized N-Cre and mCherry for Human are shown in Fig.9 for transfection into Vero cells, and the codon-optimized N-Cre and mCherry for mosquito are shown in Fig.10 for transfection into C6/36 cells. (Parts link)

Fig. 9: Backbone, insert(H) containing codon-optimized genes for Human, Product(H). Markers are lambda/Hind III.

Fig.10 : Insert(m) and Product(m) containing codon-optimized genes for Backbone and Mosquito. λ/Hind III was used for markers.

(3) Transfection into cells Two methods of transfection into cells were tested. Lipofection and electroporation. In electroporation, cells were treated with restriction enzymes on one cut of the plasmid to linearize the plasmid before transformation in order to achieve stable transformation. Due to a combination of experimental progress, we were able to try lipofection and electroporation for Vero cells and lipofection for C6/36 cells. These transformations should allow the cells to constantly express EGFP and N-Cre, and intracellular expression of C-Cre due to SRIP infection, etc., should lead to intracellular recombination and mCherry expression. In fact, we confirmed EGFP in Vero and C6/36 cells, and in Vero cells, EGFP was confirmed by both lipofection and electroporation.

Fig.11 shows the fluorescence image at the top and the bright field image at the bottom, from left to right: negative control Vero cells, lipofection Vero cells, and electroporation Vero cells. The G418 selection of the Vero cells is still insufficient because it has not been long since the transfection. However, continued culture in G418-containing medium should yield stable transformants.

Fig.12 shows the fluorescent image of C6/36 cells (top) and the bright-field image of lipofected C6/36 cells (bottom). 3 days after transfection of C6/36 cells, transformant colonies were observed. Electroporation of the C6/36 cells will begin after the wiki freeze, and we are very hopeful that it will be completed in time for the Grand Jamboree. Also, if these cells are infected with SRIP or transformed with a plasmid that expresses C-Cre intracellularly, Split-Cre will associate intracellularly and the Cre-LoxP system will cause intracellular gene recombination and mCherry expression.

Fig. 11: (A) Vero cells negative control fluorescent image, (B) Vero cells negative control bright-field image, (C) Lipofected vero cells fluorescent image, (D) Lipofected vero cells bright-field image, (E) Electroporated vero cells fluorescent image, (F) Electroporated vero cells bright-field image

Fig. 12: (A) C6/36 cells negative control fluorescent image, (B) C6/36 cells negative control bright-field image, (C) Lipofected C6/36 cells fluorescent image, (D) Lipofected C6/36 cells bright-field image

3. SRIP infection

Single-round infectious particle (SRIP) infection experiments have not been conducted because the production of SRIP and SRIP detecting cells have not been completed. However, when both are produced, infection experiments can be performed to confirm the function of the Split-Cre and Cre-LoxP systems. We can also use commercially available antibodies to determine the extent to which SRIP infection can be blocked by neutralizing antibodies.

Discussion

In summary, the only results that can be documented in this wiki are that we are halfway through the construction of the SRIP production and are one step closer to completion of the SRIP detecting cell production. The SRIP we are producing this time uses the C-Cre of Split-Cre as the inducer, which is shorter than the sequence length of the fluorescent protein used in 2018 and close to the sequence length of the Nano-Luciferase used by Dr. Suzuki, so normal SRIP should be produced. In addition, both Split-Cre have the nuclear localization signal NLS, and we used one that showed sufficient restoration of activity by reconstitution. Furthermore, since C-Cre encoded by the SRIP genome is expressed intracellularly by the

CMV promoter and N-Cre by the CAG promoter in the cell, Cre recombinase is sufficiently reconstituted to replace EGFP by the Cre-LoxP system mCherry instead of EGFP by the Cre-LoxP system. If these SRIPs and SRIP detecting cells can be used for neutralizing antibody assays, they will have a significant advantage over other methods. Integrated Human Practice

References
[1] Yamanaka A, Matsuda M, Okabayashi T, Pitaksajjakul P, Ramasoota P, Saito K, Fukasawa M, Hanada K, Matsuura T, Muramatsu M, Shioda T, Suzuki R. Seroprevalence of Flavivirus Neutralizing Antibodies in Thailand by High-Throughput Neutralization Assay: Endemic Circulation of Zika Virus before 2012. mSphere. 2021 Aug 25;6(4):e0033921. doi: 10.1128/mSphere.00339-21. Epub 2021 Jul 14. PMID: 34259560; PMCID: PMC8386448
[2] Rajaee M, Ow DW. A new location to split Cre recombinase for protein fragment complementation. Plant Biotechnol J. 2017 Nov;15(11):1420-1428. doi: 10.1111/pbi.12726. Epub 2017 Apr 20. PMID: 28317293; PMCID: PMC5633763.