Difference between revisions of "Part:BBa K2865000"

Latest revision as of 14:47, 17 October 2018

BNP promoter, heart failure inducible

This part encodes for the approximal region (-408/+100bp) of the human brain natriuretic peptide (hBNP) promoter. The use of this part is to express therapeutic genes specifically in failing cardiomyocytes, so as to enhance the selectivity of gene therapy of heart failure and reduce its side effects.

Usage and Biology

Brain natriuretic peptide (BNP), also known as B-type natriuretic peptide, is a hormone secreted by cardiomyocytes in the heart ventricles. It has been regarded as an important biomarker in diagnosis of heart failure (HF), for its level is highly related to the severity of HF. The activity of BNP promoter remains low in normal hearts and elevates dramatically in that of patients with HF. Based on the qualities mentioned above, it is natural to think that we can utilize BNP promoter to control gene expression in HF gene therapy. According to previous studies, BNP promoter can be activated by a number of factors associated with HF, including endothelin (ET), angiotensin II (Ang II), mechanical strain, and hypoxia, among others[1]. These regulating activities could be attributed to the combination of a number of transcriptional factors and cis-acting elements at the upstream of the promoter. However, knowledge about the actual transcriptional mechanisms responsible for their regulation and induction in heart remains elusive.

Characterization

To test the response of our promoter to heart failure, we constructed a new part (BBa_K2865008), containing BNP promoter and reporter EGFP. The plasmid was transfected into H9C2 rat cardiomyoblast cell line, and the intensity of fluorescence was observed under fluorescent microscope. The BNP promoter efficiency was compared with that of CMV promoter by transfecting EGFP reporter driven by the CMV promoter (BBa_K1119006) and terminated by SV40 polyA signal. Since there are various factors that could induce the promoter, we selected two widely reported types, ET-1 and AngⅡ, to stimulate expression in vitro. H9C2 cells were cultured in 24-well plates and divided into 9 groups, three of them transfected with CMV plasmids, three BNP and three negative controls. And every three groups were treated with ET-1, AngⅡ, and no reagents, respectively. On day 2 post-transfection, the fluorescence of these groups was observed. Only CMV groups expressed EGFP, while BNP promoter showed a very low activity under basal condition. After observation, ET-1(10-7mol/L) and AngⅡ(10-7 mol/L)[2] were then added to the medium of the corresponding groups. Forty-eight hours later, cells were observed again under fluorescent microscope. However, there was still no fluorescence in BNP groups (Figure. 1A-D) as well as negative controls whether added drugs or not. This result indicated that BNP promoter didn’t show high activity after drug stimulation in vitro. The fluorescence levels of ET-1, AngⅡ, or untreated CMV groups were similar(Figure. 2A-C), which suggests that CMV promoter doesn’t have specificity in failing cardiomyocytes and may have the risk of causing side effects by over-expressing in normal cells.

Figure.1. Expression of BNP-EGFP 48 h after the administration of ET-1 and AngⅡ. (A) and (B) were cells treated with 10^-7 mol/L ET-1. (A) was taken under fluorescent microscope and (B) light microscope. (C) and (D) were cells treated with 10^-7mol/L Ang

Figure.2. Representative images are shown for (A) untreated, (B) ET-1, and (C) AngⅡinduced H9C2 cells transducted with CMV-EGFP.

Reference

[1].Sergeeva, I.A. and V.M. Christoffels, Regulation of expression of atrial and brain natriuretic peptide, biomarkers for heart development and disease. Biochim Biophys Acta, 2013. 1832(12): p. 2403-13.

[2].Carlson, C., et al., Phenotypic screening with human iPS cell-derived cardiomyocytes: HTS-compatible assays for interrogating cardiac hypertrophy. J Biomol Screen, 2013. 18(10): p. 1203-11.

Sequence and Features