Great Deal! Get Instant $25 FREE in Account on First Order + 10% Cashback on Every Order Order Now

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...

1 answer below »
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 XXXXXXXXXX4µL
pLx304-cdh11-(EC2-EC5)-EGFP-2XFLAG(Vector,0.2886µg/µL XXXXXXXXXX1µL
pLx304-cdh11-(EC3-EC5)-EGFP-2XFLAG (Vector,0.4237 µg/µL) XXXXXXXXXX1µL
pLx304-cdh11-(EC4-EC5)-EGFP-2XFLAG (Vector,0.4849 µg/µL) XXXXXXXXXX1µL
pLx304-cdh11- (EC XXXXXXXXXXEGFP-2XFLAG (Vector,0.5863 µg/µL) XXXXXXXXXX1µL
pLx304-cdh11- (TM) -EGFP-2XFLAG (Vector,0.1701 µg/µL) XXXXXXXXXX1µL
BstBI (Bsp119I) XXXXXXXXXX1µL 1µL 1µL 1µL 1µL 1µL
10 X Cut Smart Buffer XXXXXXXXXX2µL 2µL 2µL 2µL 2µL 2µL
Sterile water XXXXXXXXXX13µL 15µL 15µL 15µL 15µL 15µL
iSAP XXXXXXXXXX0µL 1µL 1µL 1µL 1µL 1µL
Digestion=37ºC, 30 minutes
The digested products were separated in a 1% agarose gel/TAE Buffe
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
    Vecto
    2µL
    Insect
    10µL
     10 X T4 Ligase
    2µL
    T4 Ligase
    1µL
    Sterile wate
    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-Aga
Ca
plate (37ºC,19hr).
Nano Drop:
Abso
ance measurements made on a spectrophotometer, including any Thermo Scientific Nano Drop Spectrophotometer, will include the abso
ance of all molecules in the sample that abso
at the wavelength of interest. Since nucleotides, RNA, ssDNA, and dsDNA all abso
at 260 nm, they will contribute to the total abso
ance 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
· Pou
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 XXXXXXXXXX1 µL
· BstBI(119I) XXXXXXXXXX0.5 µL
· MluI XXXXXXXXXX0.5 µL XXXXXXXXXXfor 30 Minutes at 37ºC
· 10X Fast Digest Buffer XXXXXXXXXX µL
· Sterile water XXXXXXXXXX16 µL
The digested samples were separated in a 1% agarose gel / TAE buffer / Ethidium Bromide solution (100V, 25 Minutes).
Lane : XXXXXXXXXX XXXXXXXXXX XXXXXXXXXX XXXXXXXXXX XXXXXXXXXX XXXXXXXXXX18
Samples: 4
Answered Same DayNov 19, 2021

Solution

Pragnya answered on Nov 23 2021
62 Votes
Role of cadherin mediated signaling 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. As cadherin is known to be involved in cell growth, cellular differentiation and cell migration, this study will emphasize the specific cadherin functional activity during towards the differentiation of mesenchymal stem cells to smooth muscle cell. In this review, we focus on the role of cadherin mediated signaling 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 signaling affects lineage specification may affect 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:
Cadherins belong to the types of cell adhesion molecules (CAM) and help in cellular differentiation, proliferation and apoptosis by regulating intercellular adhesion. These cadherin are of different types according to their origin and functional sites. The cadherin molecules mediate the cellular adhesion in the presence of calcium molecules and the adhesion between cells is known as adherens junction (AJ). Studies suggests that cadherin are affected by the soluble growth factors and they activate biochemical pathways. 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. CDH11 is found in osteoblasts while CDH2 in neuronal cells. Their site of expression also suggest that CDH11 is involved in the smooth muscle cell formation. 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 was first done by adding the desired gene segment to the plasmid. The plasmid was then incubated along with the bacteria, which led to the transformation of plasmid DNA to the bacterial and expression of the required DNA fragments. The generation of pLx304—cdh11 -- EC2-EC5—GGGS—EGFP 2X FLAG which is the segment of CDH11, was done by digesting the expressed DNA. After transformation and digestion of the segment, the digested products were gel run for extraction using E.Z.N.A Gel extraction kit for isolation of DNA fragments. The digested products, which were eluted in pre-heat water, were then ligated using T4 Ligase. The transformation of the ligated segments was done by adding it to STBL3 E. coli strain used for the cloning of the DNA fragment. The sample and bacteria were incubated at required temperature, heat shocked and then inoculated on an LB agar plate to grow. A total number of 3 colonies were selected from the Agar media and the plasmid DNA was isolated using NucleoSpin mini kit by Macherey-Nagel. The isolated samples were eluted in water.
The extracted plasmid DNA segment was digested and ligated. EC2-1, EC3-1, EC5-1, ECTM-3 were sent for sequencing with primer MluI-EGFP-rev. The result showed positive clones with the MluI-EGFP-rev primer for all except TM-3. TM2 was again sent for sequencing which then showed positive result. 12 more clones were sequenced as E4-E5 did not have positive clone.
The procedure in detailed is provided below:
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...
SOLUTION.PDF

Answer To This Question Is Available To Download

Related Questions & Answers

More Questions »

Submit New Assignment

Copy and Paste Your Assignment Here