Part:BBa_K2865009
BNP-AR185-Poly(A)
AR185 linked with eGFP by Tr2 is attached to the PloyA under control of BNP Promotor(BB...).The use of this part is to express AR185 under control of BNP Promotor induced by some factors. And to certify its potential efficacy on heart failure gene therapy.
Usage and Biology
Respective induction of parts
Brain natriuretic peptide (BNP), also known as B-type natriuretic peptide, is a hormone secreted by cardiomyocytes in the heart ventricles. The testing of BNP to standard clinical assessment has been considered as an accurate and efficient diagnosis and prognostication of HF, and the use of BNP may be a potential targeting to improve clinical outcomes. [1][2][3] Circulating levels of BNP are normally very low in healthy individuals. And BNP level correlate well with ventricular wall stress and severity of HF, that is to say, BNP promoter activity is related to the severity of HF [4]. 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. However, despite decades of research, the mechanisms regulating the BNP genes during development and disease are not well understood. 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. These regulating activities could be attributed to the combination of a large variety of transcriptional factors and cis-acting elements at the upstream of the promoter. AR185 nanobodies which specifically bind to RyR2 in rat cardiomyocytes and have the ability to inhibit PKA dependent S2808 phosphorylation in vitro (detailed showing in BBa_K2865001).
Experimental Setup
This part is meant to express the AR185 gene under control of BNP promoter.AR185 is a protein monomer which can conjuge with s2808 site on RyR2 receptor to inhibit its phosphorylation. This protein is transported as an intracellular antibody. To characterize AR185 in vivo, we used ET-1, one stimulus factor, to induce BNP promoter expression. Then, the later sequence of the part, AR185, also can be expressed. We created two E. coli strains. One carried the AR185 gene that codes for AR185 under control of the BNP promoter (pSB1C3 plasmid BBa_K2865009), whereas a second strain carried an additional second plasmid with the CMV promoter (pSB1C3 plasmid;BBa_K2865010). To evaluate the potential use of anti-phosphorylation agents targeting RyR2 for the treatment of heart failure, an adeno-associated virus (AAV) based intracellular antibody delivery strategy were adopt to achieve cardiac-specific gene-therapy and demonstrated therapeutic effect both in cell-based assays and in vivo models.
Cloning
PCR Amplification
The concentration of each of the 6 parts was increased using PCR amplification.
Gel Extraction
After PCR amplification, gel extraction was carried out to ensure that the parts were correct, and to separate them.
Transformation
Transformation was carried out according to standard protocol using competent E. Coli Top 10 cells.
Sample Preparation
Liquid-Liquid Extraction using Ethyl Acetate was carried out according to standard protocols 4 hours after incubation with cholesterol.
Characterization
Fluorescence assay
To be able to ensure that AR185 is expressed, we added a gene encoding for eGFP (BBa_I13504) under induction of the same promoter (BBa_K1583112)(BNP的) to check that after lingking with another part, the promoter still works.
Therefore, a maximum eGFP of 0.007 mg/ml within 4 hours of substrate addition was observed! This was approximately 17.5 times greater than the amount quantified in past research [4]. Furthermore, this is possibly the first time Human FDX1 gene, Human FDXR gene, and Human CYP11A1 were expressed together in E Coli., as no literature on this particular combination of genes being expressed by E. Coli. was found. (长得最好的实验条件)
References
1. Maisel AS, Krishnaswamy P, Nowak RM, McCord J, Hollander JE, Duc P, Omland T, Storrow AB, Abraham WT, Wu AH, Clopton P, Steg PG, Westheim A, Knudsen CW, Perez A, Kazanegra R, Herrmann HC, McCullough PA. Rapid measurement of B-type natriuretic peptide in the emergency diagnosis of heart failure. N Engl J Med. 2002;347:161–167.
2. Januzzi JL Jr, Peacock WF, Maisel AS, Chae CU, Jesse RL, Baggish AL, O’Donoghue M, Sakhuja R, Chen AA, van Kimmenade RR, Lewandrowski KB, Lloyd-Jones DM, Wu AH. Measurement of the interleukin family member ST2 in patients with acute dyspnea: resultsfromthePRIDE(Pro-BrainNatriuretic Peptide Investigation of Dyspnea in the Emergency Department) study. J Am Coll Cardiol. 2007;50:607–613.
3. Felker GM, Hasselblad V, Hernandez AF, O’Connor CM. Biomarker-guided therapy in chronic heart failure: a meta-analysis of randomized controlled trials. Am Heart J. 2009; 158:422–430.
4. Januzzi JL Jr, Camargo CA, Anwaruddin S, Baggish AL, Chen AA, Krauser DG, Tung R, Cameron R, Nagurney JT, Chae CU, Lloyd-Jones DM, Brown DF, ForanMelanson S, Sluss PM, Lee-Lewandrowski E, Lewandrowski KB. The N-terminal Pro-BNP Investigation of Dyspnea in the Emergency department (PRIDE) study. Am J Cardiol. 2005;95:948–954.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 671
Illegal NgoMIV site found at 818
Illegal NgoMIV site found at 1753 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 242
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