So I already have written the report. I just want you to explain each section bit more and write me a conclusion based on the stuff ive written already.
Role of cadherin mediated signalling in development and stem cell differentiation, with emphasis on cadherin-11 (CDH11) (OB-cadherin). Abstract: Accumulating evidence suggests that mechanical and biochemical signals from cell-cell adhesion are critical to the specification of the lineage of stem cells. In this review, we focus on the role of cadherin mediated signalling in the development and differentiation of stem cells, focusing on two well-known cadherins, cadherin-2 (CDH2) (N-cadherin) and cadherin-11 (CDH11) (OB-cadherin). We summarize existing knowledge of the role of CDH2 and CDH11 during in vivo and in vitro development and differentiation. We also discuss engineering strategies for controlling stem cell fate decisions by using surface chemistry and micro topology to fine-tune the extent of cell adhesion. Novel strategies that enable monitoring of stem cell specification in real time can greatly facilitate these studies. We expect a better understanding of how intercellular adhesion signalling affects lineage specification may impact biomaterial and scaffold design to control stem cell fate decisions in a three-dimensional context with potential implications for tissue engineering and regenerative medicine. Introduction: Although soluble factors such as the transformation of growth factor β1 (TGF-β1), induce the differentiation of mesenchymal stem cells (MSC) towards the lineage of smooth muscle cells (SMC), the role of adherent junctions in this process is not well understood. In this study, we found that for MSC differentiation into SMCs, cadherin-11 but not cadherin-2 was needed. regulated TGF-β1 expression and affected SMC differentiation by a pathway dependent on TGF-β receptor II (TGFβRII) but independent of SMAD2 or SMAD3. Furthermore, through the Rho-associated protein kinase (ROCK) pathway, cadherin-11 activated serum response factor (SRF) and SMC protein expression. engagement increased its own expression through SRF, indicating the presence of an autoregulatory feedback loop committing MSCs to the fate of the SMC. Notably, cadherin-11-null (Cdh11(-/-)) SMC-containing tissues (such as aorta and bladder) mice showed significantly reduced SMC protein levels and decreased contractility compared to controls. This is the first report involving cadherin-11 in both in vitro and in vivo differentiation of SMC and contractile function. Method and Procedure: Transformation: · Incubate the bacteria on ice for 30 min. – 6TBL3 · Incubate the Plasmids with bacteria on ice for another 30 min. · Heat shock for 42 seconds at 42ºC. · Let it sit on ice for 2 minutes. · Add 200µL of S.O.C. medium and put it in the incubator shaker at 250 RPM,37ºC for 1 hr. · Then spread it on the agar plate. Generation of pLx304—cdh11 -- EC2-EC5—GGGS—EGFP 2X FLAG Digestion pLx304-cdh11-GGGS-EGFP-2XFLAG (Insect)(0.29µg/µL) 4µL pLx304-cdh11-(EC2-EC5)-EGFP-2XFLAG(Vector,0.2886µg/µL) 1µL pLx304-cdh11-(EC3-EC5)-EGFP-2XFLAG (Vector,0.4237 µg/µL) 1µL pLx304-cdh11-(EC4-EC5)-EGFP-2XFLAG (Vector,0.4849 µg/µL) 1µL pLx304-cdh11- (EC5) -EGFP-2XFLAG (Vector,0.5863 µg/µL) 1µL pLx304-cdh11- (TM) -EGFP-2XFLAG (Vector,0.1701 µg/µL) 1µL BstBI (Bsp119I) 1µL 1µL 1µL 1µL 1µL 1µL 10 X Cut Smart Buffer 2µL 2µL 2µL 2µL 2µL 2µL Sterile water 13µL 15µL 15µL 15µL 15µL 15µL iSAP 0µL 1µL 1µL 1µL 1µL 1µL Digestion=37ºC, 30 minutes The digested products were separated in a 1% agarose gel/TAE Buffer/Ethidium Bromide (100V,25min). Digested samples were gel extracted (E.Z.N.A gel extraction kit) and eluted in 50µL pre heat water. LIGATION-25Min, RRTRT Vector 2µL Insect 10µL 10 X T4 Ligase 2µL T4 Ligase 1µL Sterile water 5µL TRANSFORMATION: Ligated sample(5µL) was added to 50 µL STBL3 and left on ice for 30 min. Samples were heat shocked (42ºC,45 sec, ice,2min). S.O.C. Medium was added and sample was inoculated at 250 RPM,37ºC for 1 hr. Sample (200 µL) was spread on LB-Agar/Carb plate (37ºC,19hr). Nano Drop: Absorbance measurements made on a spectrophotometer, including any Thermo Scientific Nano Drop Spectrophotometer, will include the absorbance of all molecules in the sample that absorb at the wavelength of interest. Since nucleotides, RNA, ssDNA, and dsDNA all absorb at 260 nm, they will contribute to the total absorbance of the sample. Therefore, to ensure accurate results when using a Nano Drop Spectrophotometer, nucleic acid samples will require purification prior to measurement. · 1µL NFW · Apply 1µL NFW directly on the platform. · Apply 1µL NFW directly on the HP. · Blank it. · Load your sample of 1µL on the top of the HP. · Concentration: 1µg/µL · DNA/Protein Ratio-1.96 · DNA/RNA-0.04 · Concentration: 1.781µg/µL · DNA/Protein Ratio-2.28 · DNA/RNA-1.93 For Gel: · V = 50 mL · Agarose = 1% Gel, 0.5 g · Microwave for 1 min · Wait until it cools down and 2µL of Ethereum Bromide · Wait till milky colour appears · Pour Experimental Design: We found thatCadherin-11 (CDH11) regulates the synthesis of collagen and elastin, both affecting animal tissue's mechanical properties and contractile function. Using a Cdh11-null mouse model, we observed a significant decrease in the mechanical properties of Cdh11(-/-) [ Youngs ' module and ultimate tensile strength (UTS)] compared to wild mouse tissue (WT) such as aorta, bladder and skin. The deterioration of mechanical properties (Youngs ' module and UTS) has been accompanied by reduced content of collagen and elastin in Cdh11(-/-) mouse tissues as well as in culture cells. Similarly, the abolition of CDH11 in human cells abolished collagen and elastin synthesis, thus reducing their ability to generate strength. In contrast, CDH11 engagement through homophilic interactions led to rapid activation of the TGF-β and ROCK pathways as evidenced by downstream effector phosphorylation. Activation of key transcription factors, MRTF-A (also known as MKL1) and MYOCD led to significant collagen and elastin gene upregulation. Taken together, our results show a novel role for adherents in regulating extracellular matrix (ECM) synthesis with implications for many important biological processes, including tissue maintenance. SCREENING: · Plasmid - 1 µL · BstBI(119I) - 0.5 µL · MluI - 0.5 µL for 30 Minutes at 37ºC · 10X Fast Digest Buffer - 2 µL · Sterile water - 16 µL The digested samples were separated in a 1% agarose gel / TAE buffer / Ethidium Bromide solution (100V, 25 Minutes). Lane : 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Samples: 4