The rb7 matrix attachment region increases the likelihood and magnitude of transgene expression in tobacco cells: a flow cytometric study

Abstract

Many studies in both plant and animal systems have shown that matrix attachment regions (MARs) can increase expression of transgenes in whole organisms or cells in culture. Because histochemical assays often indicate variegated transgene expression, a question arises: Do MARs increase transgene expression by increasing the percentage of cells expressing the transgene (likelihood), by increasing the level of expression in expressing cells (magnitude), or both? To address this question, we used flow cytometry to measure green fluorescent protein (GFP) expression in individual tobacco (Nicotiana tabacum) cells from lines transformed by Agrobacterium tumefaciens. We conclude that MAR-mediated overall increases in transgene expression involve both likelihood and magnitude. On average, cell lines transformed with the Rb7 MAR-containing vector expressed GFP at levels 2.0- to 3.7-fold higher than controls. MAR lines had fewer nonexpressing cells than control lines (10% versus 45%), and the magnitude of GFP expression in expressing cells was greater in MAR lines by 1.9- to 2.9-fold. We also show that flow cytometry measurements on cells from isogenic lines are consistent with those from populations of independently transformed cell lines. By obviating the need to establish isogenic lines, this use of flow cytometry could greatly simplify the evaluation of MARs or other sequence elements that affect transgene expression.

Figure 1.

Schematic Diagrams of T-DNA Regions of Transformation Vectors. The right and left borders of the T-DNA are indicated with RB and LB, respectively. In the control (GFP) construct, the soluble-modified, red-shifted GFP gene is driven by the 35S promoter of Cauliflower mosaic virus with the nos polyadenylation region. The NPTII (neomycin phosphotransferase selectable marker gene) is driven by the nos promoter with the g7T polyadenylation region. An additional control construct (LGFPL) contains direct repeats of 1195 bp of λ phage spacer DNA. MGFPM contains direct repeats of the 1167-bp Rb7 MAR. Diagram schematics are not to scale.

Figure 2.

Propidium Iodide Staining of Protoplast Preparations Improves Flow Cytometry Resolution. (A) A 1-μm thick confocal section of a propidium iodide–stained protoplast preparation of a tobacco cell line uniformly expressing GFP after stable transformation with MGFPM. Intact protoplasts exclude propidium iodide (PI), whereas free nuclei and nuclei in protoplasts with compromised plasma membrane integrity stain brightly. GFP is absent from protoplasts in which nuclei stain brightly with PI. The arrow indicates a missing portion of the plasma membrane. (B) Green fluorescence histogram of wild-type protoplasts. Fluorescence was measured in relative fluorescence units (RFU). Because in wild-type cells green fluorescence intensity is below 17 RFU, transformed cells with green fluorescence below 17 RFU are considered GFP negative cells (Neg), and cells with green fluorescence above 17 RFU are considered GFP positive cells (Pos). (C) MGFPM stably transformed protoplasts stained with PI subjected to biparametric analysis of side light scatter (SSC) versus forward light scatter (FSC). Gated events inside the oval are plotted in (D). (D) Green fluorescence histogram of gated MGFPM protoplasts from (C). (E) The same data from the same MGFPM stably transformed protoplasts stained with PI were subjected to biparametric analysis of side light scatter versus relative red fluorescence. Gated events inside the oval are plotted in (F). These gated events have background levels of PI fluorescence similar to that of wild-type cells (data not shown). (F) Green fluorescence histogram of gated MGFPM protoplasts from (E). Note that after exclusion of protoplasts and debris that have high levels of PI fluorescence, the mode consistent with wild-type fluorescence was lost.

Figure 3.

Green Fluorescence Histograms of Individual Transgenic Cell Lines. Histograms from each treatment are ordered by their average green fluorescence. For simplicity, only every third histogram, ranked by average green fluorescence (lowest fluorescence, line 1; highest fluorescence, line 10) is displayed. Data shown from replicate 2 only. Green fluorescence histograms from cell lines transformed with the GFP (A), LGFPL (B), and MGFPM (C) T-DNA vectors. Note in (C), the cell line with the second lowest average green fluorescence (line 2) has a mode of cells expressing in the 300 RFU region, but the mode is partially obscured by the histograms plotted below it.

Figure 4.

Summary of GFP Expression in Individual Transgenic Cell Lines for Three Replicate Experiments. Tobacco protoplasts were analyzed by flow cytometry, and results from ∼30 cell lines each of GFP, LGFPL, and MGFPM T-DNA vector transformations are shown for each replicate. (A) Mean GFP fluorescence per cell in cell lines transformed by each of the vectors (data from both GFP positive cells and GFP negative cells are included). Error bars denote se. (B) Mean percentage of GFP negative cells in cell lines transformed by each of the vectors. Error bars denote se. (C) Box plots of GFP fluorescence per GFP positive cell in cell lines transformed by each of the vectors (data from GFP negative cells is not included). The boundaries of the boxes indicate the 25th and 75th percentiles. The whiskers above and below the box indicate the 10th and the 90th percentiles. The median and the mean are represented by a solid line within the box and a plus sign, respectively.

Figure 5.

GFP Expression Is Stable over Time in Individual Transgenic Cell Lines. Cell lines were maintained on selection for 10 months, and green fluorescence was measured by flow cytometry. Data from all cells (expressing and nonexpressing) are included. Error bars denote se.

Figure 6.

Comparison of GFP Expression in Averaged Individual Cell Lines and Population Cell Cultures. (A) Average green fluorescence histograms of all 30 individual cell lines for each treatment from the second replicate experiment (10 of which are shown in Figure 3). (B) Green fluorescence histograms from population cultures established from the same A. tumefaciens cocultivation transformation plates used to isolate individual lines summarized in (A). Three independent cultures were established for each T-DNA vector. Parameters for histograms are the same as in Figure 2B.

Figure 7.

Isolation of Callus Sectors Expressing GFP at Different Levels and Flow Cytometric Analysis. (A) A subset of calli display sectored GFP phenotype (left column). Tissue from these sectors was subcultured on fresh solid medium (right column). The plus sign denotes tissue from the sector with the higher level of green fluorescence, whereas the minus sign denotes tissue from the sector with the lower level of green fluorescence. Sectored calli from each T-DNA vector were chosen for further analysis: sample 1 from GFP T-DNA; sample 2 from MGFPM T-DNA; samples 3, 4, and 5 from LGFPL T-DNA. (B) GFP fluorescence histograms of liquid cultures initiated from sectors in the right column of (A). The black histogram is from the (−) sector, and the green histogram is from the (+) sector. Parameters for histograms are the same as in Figure 2B.

Figure 8.

Genotyping Sectors by DNA Gel Blot Analysis. DNA was isolated from liquid cultures initiated from callus sectors (Figure 6A, right column). Blots of 15 μg of DNA digested with the methylation-insensitive restriction enzyme HindIII (which cuts only once within the T-DNA) and separated by electrophoresis were probed with a portion of the right T-DNA border common to all vectors. Each banding pattern represents a transgenic fingerprint for each sector. The blot was reprobed with rDNA to account for differences in DNA loading and differences in DNA mobility attributed to possible differences in salt concentrations. Numbers to the left are approximate DNA size markers in kilobases.