To evaluate the role of split GFP as a reporter for antibody production, we constructed an overexpression vector for split GFP and antibody chains expressed simultaneously. Figure
A shows the schematic diagram representing the way the assembly of split GFP works as a reporter for the selection of antibody-producing cells. The GFP fragments, called N-GFP and C-GFP (described in the ‘Methods’ section), are co-linked with a light chain and heavy chain gene using an IRES sequence, respectively. These constructs containing an IRES sequence, pNGFP-Light and pCGFP-Heavy, led us to hypothesis that the transcription level of the light and heavy chains is highly correlated with that of N-GFP and C-GFP. From this hypothesis, our speculation extends to the idea that the high GFP-expressing clone may be the high antibody-producing clone.
Figure 1 New cell line development for high antibody-producing mammalian cells.(A) Schematic diagram for the split GFP-based cell screening method. (B) Confocal microscopic images of cells transfected with a GFP-overexpressing vector (pEGFP-C1) or bicistronic (more ...)
To confirm both the antibody expression and the assembly of the GFP with a constructed vector, a bicistronic vector containing GFP fragments and an antibody gene, pNGFP-Light and pCGFP-Heavy, was transfected into HEK293T cells. As a control, a GFP-overexpressing vector, pEGFP-C1, was also transfected into HEK293T cells. Figure
B shows the confocal microscopic images of transfected cells and antibody titer produced from them. The emission of green fluorescence from reconstituted split GFP was efficiently detected when the two fragments were co-expressed. In addition, the cells with green fluorescence could produce the antibody simultaneously. The same results were confirmed in CHO-K1 cells (data not shown). Accordingly, we proceeded with these constructs to evaluate the efficiency of this screening system in CHO cells.
To investigate whether the sorted cells by FACS are the antibody-producing CHO cells, the CHO cells with intracellular green fluorescence (First sort pool and Second sort pool) were generated by two rounds of FACS from the CHO cells co-transfected with pNGFP-Light and pCGFP-Heavy (Unsorted pool). Figure
shows the flow cytometry analysis and specific antibody productivity (qAb
) of unsorted and FACS-sorted cells. The discrimination of cells with green fluorescence was performed on a FITC/SSC diagram. The percentage of the cell population with GFP expression in the First sort pool and Second sort pool was much higher than that in the Unsorted pool. Interestingly, the increase in qAb
was highly associated with the increase in the cell population with GFP expression. Taken together, we found that the FACS-sorted cells with GFP expression are the antibody-producing CHO cells.
Figure 2 Flow cytometry analysis for green fluorescence andqAbof unsorted cells and FACS-sorted cells having reconstituted GFP. Dot plots of SSC versus log-FITC fluorescence are shown. Each dot represents a single cell; 10,000 cells are represented in each plot. (more ...)
To evaluate the efficiency of split GFP-based clone selection for high antibody-producing cells, individual clones were isolated from the Unsorted pool and the Second sort pool by the limiting dilution method. Table
shows the distribution of 116 isolated clones from the Unsorted pool and the Second sort pool with regard to antibody titer, respectively. All of the Unsorted pool-derived clones have low antibody production (less than 1
mg/L). Eighty-two out of 116 clones in the Second sorted pool have antibody production exceeding 1
mg/L. Moreover, there are four clones having over 10
mg/L in the Second sort pool-derived clones, while there are none in the Unsorted pool-derived clones. Altogether, the clone selection with split GFP-based FACS appears to be an efficient strategy to select high antibody-producing CHO cells.
Distribution of isolated clones from the Unsorted pool and the Second sort pool for antibody titer
A major issue in the development of a cell screening method using a reporter is to verify the co-relationship between the expression level of the reporter and the antibody. Especially, the screening system dealing with intracellular reporter should be evaluated because antibody is generally secreted protein. In the same context, previous reports utilizing a fluorescent protein as an intracellular reporter proved the mutual relation between productivity of desired protein and fluorescence intensity [6
To address this issue, the median GFP and qAb
of 30 selected clones were estimated. Figure
shows the relationship of the median GFP and qAb
for individual selected clones. The qAb
is highly correlated with the fluorescence intensity as indicated by the high correlation coefficient (R2
0.8947). Accordingly, we confirmed that the selected clone having high fluorescence intensity by GFP-based cell sorting is the high antibody producer with high qAb
Figure 3 Relationship between theqAband green fluorescence intensity in 30 selected clones. Median green fluorescence intensity (GFP mean) and qAb were measured by flow cytometry analysis and ELISA, respectively. The qAb and GFP mean (X, Y) pairs for each clone (more ...)
Another issue that arises with the usage of intracellular reporter is the determination of the optimum time-point for sorting and metabolic burden by co-expressing reporter gene. Unlike the use of non-split fluorescent protein, we can exclude the issue of determining the optimal point in split fluorescent protein because the split fluorescent protein can be assembled at specific conditions, such as low temperature in the case of split GFP. Previously, Bochkov and Palmenberg (2006) showed that the level of gene expression linked with attenuated IRES was about 11-fold lower than wild-type IRES [13
]. Also, the modified form of GFP with increased fluorescence intensity was reported [14
]. The introduction of attenuated IRES and improved form of GFP can be a solution to reduce the metabolic burden of fluorescent protein.
The intracellular fluorescent protein-based FACS for clone selection in rCHO cells is a reasonable strategy as it does not require complex preparation steps to capture and detect a secreted antibody. Previously, Sleiman et al. (2008) developed an efficient dual intracellular auto-fluorescent protein-associated FACS to select antibody-producing CHO cells with a high level of both antibody chains [8
]. In this system, two reporters, GFP and YFP, are applied for the heavy and light chain, respectively. In general, a correct compensation is necessary for accurate analysis when more than two colors are used as a parameter in flow cytometry [15
]. Moreover, a similar level of fluorescence intensity in GFP and YFP does not guarantee a similar expression level of the heavy chain and the light chain because fluorescence intensity is a relative value, not an absolute value. Also, the split GFP-based cell screening method developed in this study is advantageous in that one reporter is introduced for quantitative analysis. By employing this type of complementation system, we can omit the process of compensation.
The additional validation for dynamic range of the GFP assay and long-term stability of constructed cell line may be needed to successfully apply the split GFP-based cell screening method for commercial production cell line. The expanded result for dynamic range of GFP expression in high antibody-producing clones with enhanced qAb
via gene amplification system and/or cis
-acting element for augmenting gene expression may be helpful to meet the industrial standard. The genetic instability of antibody-producing CHO cell line during long-term culture in the absence of selective pressure is one of main issues in CHO cell culture [16
]. The data provided from long-term culture with constructed cell lines based on split GFP system to determine the association of GFP expression with reduced qAb
will be an interesting extension of this work.