*3.2. The E*ff*ect of TALEN Plasmids on Host Cells*

We cotransfected TALEN plasmids MCMV1-2, 3-4 or 5-6 for each well, or did not transfect any plasmids as an untransfected (negative) control, using lipofectamine. The cell number was increasing for the first 5 days, although a slight decrease was observed on the 7th day. The growth period for NIH3T3 cells is from 1 to 5 days. All the four growth curves show almost the same trend for 7 days. TALEN plasmids MCMV1-2, 3-4 and 5-6 had no obvious effect on the growth of NIH3T3 cells compared with the untransfected control (Figure 3). Additionally, similar data were available in the cell viability assay (Table 2). These results demonstrated that the growth of cells was not influenced by the TALEN plasmids MCMV1-2, 3-4 and 5-6 for the first 5 days. TALEN plasmids exhibited no significant inhibition or enhancement on host cells.

**Figure 3.** Host cell growth under the effect of TALEN plasmids. NIH 3T3 cells (1.00 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/mL) were transfected with TALEN plasmids MCMV1-2, 3-4 or 5-6, respectively. The total cells were harvested and counted 1, 3, 5 and 7 days post transfection, respectively.


**Table 2.** Cell viability assay (initial NIH 3T3 cell count: 1.00 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/mL).

The cell number is expressed by cells/mL, and all data are indicated as Mean ± SD (standard deviation).

#### *3.3. The E*ff*ects of TALEN Plasmids on MCMV Titer*

Host NIH3T3 cells were divided into two groups as follows: (1) TALEN plasmid transfection was prior to the MCMV infection by 1 day; (2) MCMV infection was prior to TALEN plasmid transfection by 1 day.

When the specific TALEN plasmid (MCMV1-2, 3-4 and 5-6) transfection was prior to the viral infection, we found that the viral titers decreased by about 65%, 75%, and 25%, 1, 3 and 5 days post infection, respectively, compared with the controls, using lipofectamine as a transfection reagent (Figure 4A–C). Additionally, the viral titers decreased by about 50%, 60%, and 25%, 1, 3 and 5 days post infection, respectively, compared with the controls, using NKS11 as a transfection reagent (Figure 4D–F).

**Figure 4.** TALEN plasmid transfection was prior to MCMV infection. When using lipofectamine as a transfection reagent, the viral titers were determined 1, 3 and 5 days post infection, respectively, in (**A**–**C**). When using NKS11 as a transfection reagent, the viral titers were determined 1, 3 and 5 days post infection, respectively, in (**D**–**F**). All the data are expressed by columns (mean ± standard deviation).

However, if the viral infection was prior to the plasmid transfection, we found that TALEN plasmids resulted in no obvious growth inhibition on MCMV at 1, 3, 5 and 7 days post transfection, compared with the controls using lipofectamine or NKS11 as a transfection reagent (Figure 5).
