Functional analysis of Drosophila HSP70 promoter with different HSE numbers in human cells

Abstract

The activation of genetic constructs including the Drosophila hsp70 promoter with four and eight HSE sequences in the regulatory region has been described in human cells. The promoter was shown to be induced at lower temperatures compared to the human hsp70 promoter. The promoter activity increased after a 60-min heat shock already at 38 °C in human cells. The promoter activation was observed 24 h after heat shock for the constructs with eight HSEs, while those with four HSEs required 48 h. After transplantation of in vitro heat-shocked transfected cells, the promoter activity could be maintained for 3 days with a gradual decline. The promoter activation was confirmed in vivo without preliminary heat shock in mouse ischemic brain foci. Controlled expression of the Gdnf gene under a Drosophila hsp70 promoter was demonstrated. This promoter with four and eight HSE sequences in the regulatory region can be proposed as a regulated promoter in genetic therapeutic systems.

Figure 1. Nucleotide sequence of the hsp70+500 bp fragment cloned into pGem-T Easy.

Capital letters indicate the hsp70 promoter sequence (453 bp). The 100 bp regulatory sequence included in the 500 bp regulatory region is shown in italics. Sequencing was performed using the reverse primer phsR (5^′-taacgaattcccaattccctattcag-3′).

Figure 2. Diagram of heat shock elements (HSEs) in the regulatory region of generated factors B7 (upper) and F7 (lower).

Figure 3. Diagram of genetic constructs containing the gfp gene under control of the hsp70 promoter with the transgenic regulatory region of (A) 100 bp (B7) or (B) 500 bp (F7).

Figure 4. Western blot analysis of HEK293 cells exposed to heat shock at 40°C using antibodies against GFP (Rabbit/polyclonal Abcam ab 290).

1, proteins isolated from HEK293 cells with gfp under control of the CMV promoter; 2–4, proteins isolated from HEK293/B7 cells; 5–6, proteins isolated from HEK293/F7 cells; 7, proteins isolated from HEK293 cells.

Figure 5. HEK293 cells after transfection with vectors B7 (A, B) and F7 (C, D).

A micrographs of cell culture 48h after heat shock at 42°C made in phase contrast (A, C) and GFP fluorescence (B, D). Scale bar on D–200 µm.

Figure 6. Heat shock effect on HEK293/B7/gfp and HEK293/F7/gfp (A) and HEK293/B7/Gdnf/gfp and HEK293/F7/Gdnf/gfp (B) cells studied by flow cytometry.

A. Flow cytometry data on HEK293/B7/gfp and HEK293/F7/gfp cells 24h and 48 h after heat shock at 40 and 42°C. The proportion of GFP-positive HEK293/F7/gfp cells 24 and 48 h after heat shock was significantly higher than the control at 37°C (p<0.05). A significant increase in the proportion of GFP-positive HEK293/B7/gfp cells was observed only 48 h after heat shock (p<0.05). Asterisk indicates significant difference between HEK293/B7/gfp and HEK293/F7/gfp cells (p<0.05). B. Flow cytometry data on HEK293 cells transfected with B7/Gdnf-gfp and F7Gdnf/gfp. The same cultures not exposed to heat shock were used as control (37°C). The proportion of GFP-positive cells 24 and 48 hours after heat shock at 40 and 42°C significantly (p<0.05) differed from the control (37°C). Data represent the mean - SD from three independent experiments.

Figure 7. Temperature dependence of Drosophila hsp70 promoter activation in HEK293/B7/gfp and HEK293/F7/gfp cells studied by flow cytometry.

Ordinate: proportion of GFP-positive cells 24 h (A) and 48 h (B) after heat shock at 38–42°C and at 37°C (control).

Figure 8. Gel electrophoresis of plasmid DNA amplified by PCR using primers Gdnf-F and gfpR.

1, 100(Termo Scientific, # SM0244); 2, B7; 3, F7; 4, B7/Gdnf; 5, F7/Gdnf.

Figure 9. Genetic constructs containing a chimeric gene encoding glial cell line-derived neurotrophic factor and green fluorescent protein under control of hsp70 promoter with different regulatory regions of (A) 100 bp (B7/Gdnf/gfp) and (B) 500 bp (F7/Gdnf/gfp).

Figure 10. GFP positive HEK293 cells after transfection with B7/Gdnf/gfp (A) and F7/Gdnf/gfp (B) 24 h after heat shock at 40°C.

Scale bar on B – 100 µm.

Figure 11

A, Flow cytometry data on HEK293/B7/Gdnf/gfp and HEK293/F7/Gdnf/gfp cells 24 (red) and 48 h (green) after heat shock at 40 (left) and 42°C (right). B, HEK293 cells transfected with F7/gdnf-gfp 24 h, three days (72 h), and five days (120 h) after heat shock at 42°C and injection into the caudate-putamen (striatum) of mouse. Frontal section of the mouse brain (40 µm) at the injection site. Scale bar -0.5 mm. C, Transgenic GFP expression in not heat-shocked HEK293/F7/gfp cells in the mouse brain three days after injection of cells and endothelin-1 (indicated by the arrow) and at a distance from the injection site (the fraimed area in A is enlarged in B). Scale bar on C is 500 µm and on D 100 µm.

Figure 12. GFP-expression in not heat-shocked HEK293/B7/gfp and HEK293/F7/gfp cells 24, 72, and 120 h after their injection into the mouse brain with and without preliminary endothelin-1 administration.

A–C, transgenic GFP expression in not heat-shocked HEK293/F7/gfp cells in the mouse brain three days after injection of cells and endothelin-1 24 h (A), three. days (B), and 5 days (C) after injection of cells and endothelin-1. Scale bar on C–0.5 mm. D, and E, - analysis of GFP-positive HEK293/B7/gfp and HEK293/F7/gfp cell counts 24, 72, and 120 h after their injection into the mouse brain without preliminary heat shock. GFP positive cells counts found on the brain sections of control animals injected with transfected cells without (C) and with (D) preliminary endothelin-1 administration. Cells were counted using fluorescent microscope on brain sections in the injection area. Cell counts on B were significantly (p<0.05) enhanced in comparison with cell counts on A. Asterisk indicates significant difference (p<0.05) between cells transfected with B7/gfp and F7/gfp.