Part:BBa_K1720003

Human phosphodiesterase 5A gene silencing device NO.1

This device is uesd for silencing the human phosphodiesterase 5A (PDE5A) gene.A U6 promoter driving a designed, synthetic shRNA-like miRNA followed by the terminator.

PDE5A is a cGMP-binding, cGMP-specific phosphodiesterase, a member of the cyclic nucleotide phosphodiesterase family. This phosphodiesterase specifically hydrolyzes cGMP to 5'-GMP. It is involved in the regulation of intracellular concentrations of cyclic nucleotides and is important for smooth muscle relaxation in the cardiovascular system.

We designed 3 silencing device and test their function at the same time.Here is the another two device :BBa_K1720004,BBaK172005

This device with a GFP reporter was then transfected into HEK293 cells by lentiviral vector.Once we silence the PDE5A gene the level of cGMP will be up regulated as a result. The positive control was HEK293 cells that treat with Sodium Nitroprusside ,a NO donator that activate sGC and up regulate the level of cGMP. A negative control was made by transfecting an empty vector that does not contain scilencing device. We used Elisa to detect cGMP level. The results are as follow:

Green fluorescence signal was observed under fluorescence microscope

cGMP concentration after treat with differenct scilencing device

cGMP concentration after treat with 10umol/L SNP for different time gradient

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 273
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 247
  
• 1000
  COMPATIBLE WITH RFC[1000]

Vector Map：

Vector Components:

Virus Titer: (3.23±2)×10^8 TU/ml

Funtional titer is determined based on q-PCR amplification of a small fragment from the lentiviral vector-WRPE that is integrated into the genome of transduced 293T cells.

Experiment 1:

At the beginning of our experiment, we aimed to prove that HEK293 cells can be transfected by our vector. In our vector we inserted EGFP gene as a repoter.Once HEK293 cells are transfected successfully green fluorescence signal will be observed under fluorescence microscope.

Protocol： 1. Seed cells to be 40% confluent at a 35mm culture dish.

2. Dilute 10ul lentiviral vector in 1ml DMEM medium containing 10% FBS

3. Withdraw culture medium from 35mm culture dish.

4. Add vector-DMEM complex to cells

5. Incubate for 15 hours.

6. Withdraw vector-DMEM complex from culture dish.

7. Add 2ml DMEM medium containing 10% FBS to cells and incubate for 10 hours

8. Observe the cells under Inverted fluorescence microscope.

Result:

File:SCUT China shRNA1

From the picture we can see that vivo green fluorescence signal was observed which indicated that HEK293 cells had been transfected successfully!

Experiment 2:

After we proved that HEK293 cells can be transfected, PDE5A gene expression levels were determined by real-time PCR.

Protocol：

Result:

Experiment 3:

After we scilencing the PDE5A gene,we used cGMP Elisa kit to detect the cGMP concentration to see whether cGMP concentration can be up regulated by our scilencing device.