SV40 intron, a potent strong intron element that effectively increases transgene expression in transfected Chinese hamster ovary cells

Among five intron elements positioned downstream of an eGFP expression cassette in a CMV promoter-driven vector, the SV40 intron produces the highest transfection efficiency in CHO-S cells (85.3%), followed by the intervening sequence (IVS) intron (73.8%), the adenovirus tripartite leader (TPL) intron (68.1%), hCMV intron A (43.2%), and the Chinese hamster EF-1α gene intron 1 (CHEF1 intron, 22.1%), measured by flow cytometry 48 hours after Lipofectamine 3000 transfection. Transfection efficiency falls off exponentially as vector size increases across these five constructs, and the SV40 intron — the smallest of the five elements at 99 nucleotides — produces both the smallest vector and the highest transfection efficiency, while the largest construct, containing CHEF1 intron, produces the lowest.

Transient eGFP expression, measured by mean fluorescence intensity 48 hours after transfection, follows the same ranking as transfection efficiency: constructs containing SV40 intron, IVS intron, and TPL intron each produce significantly higher transient eGFP expression than constructs containing hCMV intron A or CHEF1 intron (P < 0.05), with SV40 intron producing the highest transient expression overall.

After G418 selection of stably transfected CHO cells, the SV40 intron construct yields the highest proportion of high-eGFP-expressing colonies at 81.8%, ahead of hCMV intron A (76.0%), IVS intron (65.6%), and TPL intron (51.5%); the CHEF1 intron construct yields only 22.5%, significantly lower than the other four constructs (P < 0.05). CHO cells stably transfected with the SV40 intron construct also proliferate faster than cells carrying any of the other four intron constructs.

In CHO cells stably selected for 30 days, the SV40 intron increases eGFP protein expression 4.39-fold and hCMV intron A increases it 3.69-fold relative to the IVS intron construct (set to 1.0), while the TPL intron and CHEF1 intron constructs produce only 0.18-fold and 0.008-fold of the IVS intron's expression, respectively; SV40 intron and hCMV intron A each produce significantly higher stable eGFP protein expression than IVS intron, and TPL intron and CHEF1 intron produce significantly lower expression (P < 0.05). This protein-level ranking does not track eGFP mRNA abundance in the same cells: relative to IVS intron (set to 1.0), hCMV intron A produces the highest eGFP mRNA level (1.92-fold), followed by CHEF1 intron (1.78-fold), then IVS intron, SV40 intron (0.85-fold), and TPL intron (0.34-fold) — so CHEF1 intron drives higher eGFP mRNA levels than SV40 intron despite producing far less eGFP protein, indicating eGFP protein expression level in this system is not determined by eGFP transcript abundance.

Stable eGFP protein expression level also does not correlate with eGFP transgene copy number in CHO cells: the SV40 intron construct, which produces the highest eGFP protein expression, corresponds to a relatively low gene copy number as measured by qPCR relative to the IVS intron construct, while the TPL intron construct, which produces one of the lowest protein expression levels, corresponds to a relatively higher gene copy number.

The coefficient of variation in eGFP expression across a stably transfected CHO cell population is lower for the SV40 intron, hCMV intron A, IVS intron, and TPL intron constructs than for the CHEF1 intron construct, indicating more uniform per-cell transgene expression with these four introns than with CHEF1 intron in a stable CHO cell pool.

The SV40 intron and hCMV intron A were the first introns shown to enhance transgene expression when positioned downstream, rather than upstream, of an expression cassette in Chinese hamster ovary cells. By contrast, TPL intron and CHEF1 intron — which contain substantially more transcription-factor-binding motifs than SV40 intron or hCMV intron A — do not enhance eGFP expression when positioned downstream of the cassette in this system, suggesting introns with more transcription-factor-binding motifs are better suited to positioning upstream of an expression cassette rather than downstream.

When the SV40 intron is combined with an erythropoietin (EPO) expression cassette instead of the eGFP cassette, CHO cell clones grown in serum-free suspension culture secrete a mean of 68.9 ± 3.2 mg/l of EPO — a 2.72-fold increase over clones carrying the equivalent IVS intron construct — with the single highest-producing SV40 intron clone reaching 86.3 mg/l of secreted EPO; Western blot confirms higher EPO protein levels for the SV40 intron construct than for the IVS intron construct, showing that the SV40 intron's downstream-positioning benefit extends from the eGFP reporter to a secreted therapeutic protein.