iGEM 2023 — Team jiangsu-united

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2023/08/03：

ATDC5 Cell recovery and differentiation by the instructor Ms. Huang.

2023/08/09：

Ice breaking activities, team cooperation games, design of team name,slogan and logo, choose team
uniforms.

2023/08/10：

Visited the lab, prepared LB solid medium, high temperature autoclave sterilization, prepared LB solid
medium plate, cell passage, transformed and cultivated E. coli strain.

2023/08/11：

Selected and cultivated monoclonal colonies, review the experimental outline.

2023/08/12：

Extracted DNA of plasmid from bacteria solution, agarose gel electrophoresis, DNA gel extraction, cell
passage.

2023/08/13：

ATDC5 cells transformation, Alcian blue dyeing, prepared Tris-HCl solution with different pH values (for
configuring SDS-PAGE electrophoresis gel).

2023/08/14：

Add IL-1β (10 ng/mL) to the growth medium of ATDC5 cells, capture pictures of ATDC5 cells stained with
Alcian blue dye, prepared transfer buffer for western blot, prepared SDS-PAGE electrophoresis gel,
transformed and cultivated E. coli strain.

2023/08/16：

Western blot, qRT-PCR, extracted DNA of plasmid from bacteria solution, agarose gel electrophoresis, DNA
gel extraction.

2023/08/17：

Western blot, qRT-PCR.

2023/08/19：

Result Analysis: Western blot and qRT-PCR.

2023/08/20：

Team meeting.

2023/08/20：

Team meeting.

2023/08/21：

Cells were transfected with sh-1, sh-2, and sh-N plasmid, and the cells were transfected with fluid.

2023/08/22：

Add IL-1β to the growth medium of ATDC5 cells for stimulation.

2023/08/23：

Cell protein and total RNA were extracted.

The expression level of OGN protein was investigated by Western blot.

2023/08/24：

qRT-PCR: investigation of the transcriptional levels of OGN and other matrix metabolism-related genes,
including Col2, Aggrecan, Sox9, MMP3 and MMP13.

2023/08/25

Result Analysis: qRT-PCR.

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Overview

In this study, we will construct a cell model of chondrocyte matrix metabolism disorder induced by
inflammatory factor IL-1β, and use the intervention method of gene overexpression to clarify the regulation
of OGN expression in chondrocytes by inflammatory factors(Figure 1). To determine the effect of OGN on
IL-1β-regulated chondrocyte matrix metabolism.

![](https://static.igem.wiki/teams/4840/wiki/engineering-success/1.png)

Cycle 1: [BBa\_K4840002](http://parts.igem.org/Part%3ABBa_K4840002) (pCMV-3Tag-1A-OGN)

Design

We selected the glycoprotein-Osteoglycin ( OGN ), which is involved in the regulation of matrix metabolism
in various tissues. At the same time, the restriction sites EcoRI and NotI were added at both ends of the
primer. At the same time, in order to be successfully transfected into cells, we selected the plasmid vector
pCMV-3Tag-1A, digested OGN and plasmid vector pCMV-3Tag-1A, and connected OGN and plasmid vector
pCMV-3Tag-1A by T4 ligase. Finally, we constructed the overexpression plasmid of OGN , named
pCMV-3Tag-1A-OGN ( Figure 2 ).

![](https://static.igem.wiki/teams/4840/wiki/engineering-success/2.png)

Figure 2. The map of pCMV-3Tag-1A-OGN plasmid

Build

In this work, we first constructed OGN overexpressed plasmid. In brief, DNA fragment of OGN was amplified
by using the cDNA of ATDC5 cells and then inserted into the pCMV-3Tag-1A vector. The E. coli strains
solution transformed with constructed plasmid were spread onto LB solid medium plate containing
corresponding antibiotic and cultured at 37℃ overnight (Figure 3).

![](https://static.igem.wiki/teams/4840/wiki/engineering-success/3.png)

Figure 3. Monoclonal colonies of E. coli transformed with pCMV-3Tag-1A-OGN plasmid

We randomly selected several monoclonal colonies from the plate for cultivation, and extracted plasmid DNA
from the cultured bacterial solution. The oligo DNA in plasmid was identified by using agarose gel
electrophoresis (Figure 4A). Our results show that a band of about 5000 bp is present in sample 2 and 8,
suggested that pCMV-3Tag-1A-OGN plasmid were constructed successfully. At the same time, we sent the
positive monoclonal antibody to the company for sequencing. Figure 4B showed that the gene sequence was
correct and there was no mutation. It proves that pCMV-3Tag-1A-OGN plasmid was successfully constructed.

![](https://static.igem.wiki/teams/4840/wiki/engineering-success/4.png)

Figure 4. Electrophoretic gel (left) and Sequencing (right) Verification of pCMV-3Tag-1A-OGN plasmid

Test

Identification of ITS-induced ATDC5 cell

In order to explore the function of OGN on the matrix metabolism of chondrocytes. We firstly added ITS to
the growth medium to induce the differentiation of ATDC5 cells. Since mature chondrocytes contain a large
amount of proteoglycan, which can be stained blue by Alcian Blue, we performed related assays in ITS-induced
ATDC5 cells. As shown in Figure 5, our cultured ATDC5 cells have differentiated into chondrocytes.

![](https://static.igem.wiki/teams/4840/wiki/engineering-success/5.png)

Figure 5. Identification of chondrocytes by microscope

2.The increase of OGN expression improved matrix metabolism in IL-1β-treated chondrocytes

Next, we established an in vitro research model of OA by using IL-1β as stimulator on chondrocytes and
detected the mRNA and protein levels of OGN in the cells by using qRT-PCR and western blotting. As shown in
the Figure 6, the mRNA and protein levels of OGN in IL-1β-stimulated chondrocytes were significantly
decreased compared with those of control group, that is consistent with the clinical observation that OGN
expression in chondrocytes from osteoarthritis patients is reduced. Moreover, both the mRNA and protein
levels of OGN in chondrocytes transfected with previously constructed pCMV-3Tag-1A-OGN plasmid were notably
increased compared to the mock group, further indicating the overexpressed plasmid was successfully
constructed.

![](https://static.igem.wiki/teams/4840/wiki/engineering-success/6.png)

Figure 6. pCMV-3Tag-1A-OGN plasmid antagonisted IL-1β-induced inhibition on OGN expression in chondrocytes
(A) qRT-PCR results; (B) western blot results

Additionally, we investigated whether OGN participates in the pathogenesis of OA. As shown in Figure 7,
mRNA levels of matrix metabolism-related genes such as Aggrecan, Col-2 and Sox9 were downregulated, while
MMP3 and MMP13 mRNA levels were upregulated in IL-1β-induced chondrocytes. These results indicated the
disruption of matrix metabolism in chondrocytes. Notably, OGN overexpression antagonized such IL-1β-induced
effects in chondrocytes. Thus, all results suggests that OGN is a promising therapeutic target for OA.

![](https://static.igem.wiki/teams/4840/wiki/engineering-success/7.png)

Figure 7. OGN overexpression antagonisted IL-1β-induced matrix metabolism disorder in chondrocytes

Learn

All results suggests that OGN is a promising therapeutic target for OA. In the future, we can optimize the
vector of OGN. First, OGN plasmid cannot be directly used to overexpress OGN in chondrocytes in vivo. It is
necessary to find a suitable delivery vector to carry it into cells. Second, we find a suitable chondrocyte
targeting carrier to make OGN highly expressed in chondrocytes.In addition, it can further study the
pathogenesis of OA and explore new treatment strategies for OA by using new drug targets of OGN, which has
important clinical significance and social value.

Cycle2: [BBa\_K4840008](http://parts.igem.org/Part%3ABBa_K4840008) ~
[BBa\_K4840010](http://parts.igem.org/Part%3ABBa_K4840010)

(RNAi-ReadypSIREN-RetroQ--ShRNA-1;RNAi-ReadypSIREN-RetroQ-ShRNA-2; RNAi-Ready
pSIREN-RetroQ-ShRNA-N)

Design

We selected the glycoprotein-Osteoglycin (OGN)，and further constructed knockout plasmid of OGN. At the same
time, in order to be successfully transfected into cells, we selected the plasmid vector
RNAi-ReadypSIREN-RetroQ. We were designed two interference sequences (ShRNA-1, ShRNA-2 ) and a negative
control sequence (ShRNA-N ) by Ambion siRNA Target Finder. At the same time, the restriction endonuclease
BamHI and EcoRI sites were designed at both ends of the sequence. The RNAi-ReadypSIREN-RetroQ and ShRNA-1,
ShRNA-2 , ShRNA-N were ligated using T4 ligase, respectively named RNAi-ReadypSIREN-RetroQ--ShRNA-1;
RNAi-ReadypSIREN-RetroQ-ShRNA-2; RNAi-Ready pSIREN-RetroQ-ShRNA-N ( Figure 8 ABC).

![](https://static.igem.wiki/teams/4840/wiki/engineering-success/8-1.png)
![](https://static.igem.wiki/teams/4840/wiki/engineering-success/8-2.png)
![](https://static.igem.wiki/teams/4840/wiki/engineering-success/8-3.png)

Figure 8 . The map of RNAi-ReadypSIREN-RetroQ--ShRNA-1; RNAi-ReadypSIREN-RetroQ-ShRNA-2; RNAi-Ready
pSIREN-RetroQ-ShRNA-N

Build

We designed and synthesized two interference sequences of OGN gene for the construction of OGN gene silencing
plasmids and performed similar experiments to explore whether the OGN is involved in the initiation and
progression of OA.

Briefly, the forward and reverse sequences of ShRNA were connected by annealing. The obtained DNA fragments
was inserted into the RNAi-Ready pSIREN-RetroQ vector. The E. coli strains solution transformed with
constructed plasmid were spread onto LB solid medium plate containing corresponding antibiotic and cultured at
37℃ overnight (Figure 9).

![](https://static.igem.wiki/teams/4840/wiki/engineering-success/9.png)

Figure 9. Monoclonal colonies of E. coli transformed with OGN gene silencing plasmids (A) RNAi-Ready
pSIREN-RetroQ-ShRNA-1; (B) RNAi-Ready pSIREN-RetroQ-ShRNA-2; (C) RNAi-Ready pSIREN-RetroQ-ShRNA-N

We randomly selected several monoclonal colonies from the plate for cultivation, and extracted plasmid DNA
from the cultured bacterial solution. The oligo DNA in plasmid was identified by using agarose gel
electrophoresis (Figure 10A). At the same time, we sent the positive monoclonal antibody to the company for
sequencing. Figure 10 B. C.D showed that the gene sequence was correct and there was no mutation.

It proves that RNAi-Ready pSIREN-RetroQ-ShRNA-1, RNAi-Ready pSIREN-RetroQ-ShRNA-2, RNAi-Ready
pSIREN-RetroQ-ShRNA-N plasmid were successfully constructed.

![](https://static.igem.wiki/teams/4840/wiki/engineering-success/10.png)

Figure 10. Electrophoretic gel (A) and Sequencing (B.C.D) Verification of RNAi-Ready pSIREN-RetroQ plasmid

Test

1.OGN gene silencing can induce matrix metabolism disorder in chondrocytes

As shown in Figure 11 and Figure 12, the mRNA and protein levels of OGN were significantly decreased in
IL-1βgroup and OGN-Sh-1, 2 group compared with those of Control group or OGN-Sh-N group. In addition, the
results of qRT-PCR revealed that the decrease of OGN expression can induce the disruption of matrix metabolism
in chondrocytes, indicative of the regulatory role of OGN in OA.

![](https://static.igem.wiki/teams/4840/wiki/engineering-success/11.png)

Figure 11. RNAi-Ready pSIREN-RetroQ-ShRNA-1 and 2 plasmids inhibited the expression of OGN in chondrocytes
(A) qRT-PCR results; (B) western blot results

![](https://static.igem.wiki/teams/4840/wiki/engineering-success/12.png)

Figure 12. The decrease of OGN expression induced matrix metabolism disorder in chondrocytes

Learn

OGN-Sh-1, and OGN-Sh-2 gene silencing have a significant effect on OGN. In the future, we can design more
gene silencing sequences to further study the effect of different silencing sequences on OGN expression and
try to find the optimal silencing sequence. In order to clarify whether OGN is involved in the regulation of
IL-1β on chondrocyte ECM metabolism ; verify the potential application of OGN as a functional target for
relieving OA and provide data support. It provides new ideas for curing arthritis with high morbidity and high
disability rate, and brings huge medical care burden and economic burden to the whole society.

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Total RNA extraction and reverse transcription

1) Materials

|  |  |
| --- | --- |
| **Reagents** | **Apparatus** |
| TRIzol | Micro-centrifuge tube (1.5 mL) |
| Chloroform | Pipette (range: 1 μL-10 μL) |
| Isopropanol | Pipette (range: 0.1 mL-1 mL) |
| Ethanol (75%) | Centrifuge |
| 5× HiScript III RT SuperMix | Spectrophotometer |
| DNase/RNase-free water | Thermostat |

2) Procedure

Ø Total RNA extraction

u Remove cell growth media.

u Add 1 mL of TRIzol reagent directly to the cell culture
plate to lyse the cells.

u Pipet the lysate up and down several times to homogenize.

u Incubate for 5 min to permit complete dissociation of the
nucleoproteins complex.

u Transfer the lysate to a tube.

u Add 0.2 mL of chloroform, then securely cap the tube.

u Upside down a few times, then incubate for 5 min at room
temperature.

u Centrifuge the sample for 15 min at 12,000 × g at
4℃.

u Transfer the colorless upper aqueous phase to a new tube.

u Add 0.5 mL of isopropanol, then incubate for 10 min at room
temperature.

u Centrifuge the sample for 10 min at 12,000 × g at
4℃.

u Remove the the supernatant.

u Add 1 mL of 75% Ethanol to the tube and pipet up and down
several times.

u Centrifuge the sample for 10 min at 12,000 × g at
4℃.

u Remove the the supernatant and open the lid of tube.

u Dry for few minutes at room temperature.

u Dissolve RNA with 20 μL of DNase/RNase-free water.

u Detect RNA concentration by using a spectrophotometer.

Ø Reverse transcription

u Prepare reaction solution.

|  |  |
| --- | --- |
| **Reagents** | **Volume/mass** |
| Total RNA | 1 μg |
| 5× HiScript III RT SuperMix | 4 μL |
| DNase/RNase-free water | to 20 μL |

u Set up reaction program.

|  |  |  |
| --- | --- | --- |
| Step 1 | 37℃ | 15 min |
| Step 2 | 85℃ | 5 s |
| Step 3 | 10℃ | ∞ |

u Start the program.

Total RNA extraction and reverse transcription

1) Materials

|  |  |
| --- | --- |
| **Reagents** | **Apparatus** |
| Template cDNA | PCR tube (0.2 mL) |
| Forward primer (20 μM) \* | Pipette (range: 1 μL-10 μL) |
| Reverse primer (20 μM) \* | Pipette (range: 1 μL-100 μL) |
| 2× Rapid Taq Master Mix | Centrifuge |
| DNase/RNase-free water | Thermostat |

2) Procedure

Ø Prepare reaction solution.

|  |  |
| --- | --- |
| **Reagents** | **Volume** |
| Template cDNA | 5 μL |
| Forward primer (20 μM) \* | 1 μL |
| Reverse primer (20 μM) \* | 1 μL |
| 2× Rapid Taq Master Mix | 25 μL |
| DNase/RNase-free water | to 50 μL |

Ø Set up reaction program.

|  |  |  |
| --- | --- | --- |
| Step 1 | 98℃ | 10 s |
| Step 2 | 55℃ | 15 s |
| Step 3 | 72℃ | 60 s |
| Step 4 | Goto step 1, 30 more times | |
| Step 5 | 4℃ | ∞ |

Ø Start the program.

\* Sequence of primers：

|  |  |  |
| --- | --- | --- |
| **Gene** | **Forward primer** | **Reverse primer** |
| OGN | GAATTCATGGAGACTGTGCACTCTA | CTCGAGTTAGAAGTATGACCCTA |

Overlap extension PCR (OE PCR)

1) Materials

|  |  |
| --- | --- |
| **Reagents** | **Apparatus** |
| ShRNA-1/-2/-N sequences | PCR tube (0.2 mL) |
| 5× Annealing buffer for DNA oligos | Pipette (range: 1 μL-10 μL) |
|  | Centrifuge |
|  | Thermostat |

2) Procedure

Ø Prepare reaction solution.

|  |  |
| --- | --- |
| **Reagents** | **Volume** |
| ShRNA-1/-2/-N forward sequences (20 μM) \* | 10 μL |
| ShRNA-1/-2/-N reverse sequences (20 μM) \* | 10 μL |
| 5× Annealing buffer for DNA oligos | 5 μL |

Ø Set up reaction program.

|  |  |  |
| --- | --- | --- |
| Step 1 | 95℃ | 2 min |
| Step 2 | decrement 0.1℃ every 8 s | |
| Step 3 | Goto step 2, 700 more times | |
| Step 4 | 4℃ | ∞ |

Ø Start the program.

\* Sequence of ShRNA：

|  |  |  |
| --- | --- | --- |
| **ShRNA** | **Forward** | **Reverse** |
| ShRNA-1 | 5’-GATCCGATGCCCACATGC CTGTTGTCTGTGAAGCCACAGATGGGACA ACAGGCATGTGGGCATCTTTTTTG-3’ | 5’-GATCCGATGCCCACATGCCT GTTGTCTGTGAAGCCACAGATGGGACAAC AGGCATGTGGGCATCTTTTTTG-3’ |
| ShRNA-2 | 5’-GATCCGTCAGCTTATCTTTATG CACCTGTGAAGCCACAGATGGGGTGCATAAAG ATAAGCTGACTTTTTTG-3’ | 5’-AATTCAAAAAAGTCAGCTT ATCTTTATGCACCCCATCTGTGGCTTCACAG GTGCATAAAGATAAGCTGACG -3’ |
| ShRNA-N | 5’-GATCCGCCAGA CCTGCAACCGATATCTGTGAAGCCACAGA TGGGATATCGGTTGCAGGTCTGGCTTTTTTG-3’ | 5’-AATTCAAAAAAGCCA GACCTGCAACCGATATCCCATCTGTGGCT TCACAGATATCGGTTGCAGGTCTGGCG -3’ |

Restriction enzyme double digestion

1) Materials

|  |  |
| --- | --- |
| **Reagents** | **Apparatus** |
| Vector or OGN fragment | Micro-centrifuge tube (1.5 mL) |
| CutSmart | Pipette (range: 1 μL-10 μL) |
| EcoRI enzyme | Pipette (range: 1 μL-100 μL) |
| XhoI enzyme | Centrifuge |
| ddH2O | Thermostat |

2) Procedure

Ø Prepare reaction solution in tubes.

|  |  |
| --- | --- |
| **Reagents** | **Volume/mass** |
| Vector or OGN fragment | 1 μg |
| CutSmart | 5 μL |
| EcoRI enzyme | 1 μL |
| XhoI enzyme | 1 μL |
| ddH2O | to 50 μL |

Ø Centrifuge the liquid to the bottom of the tubes.

Ø Place the tubes in the thermostat at 37℃ for 15 min and 65℃ for
20 min.

Ligation reaction

1) Materials

|  |  |
| --- | --- |
| **Reagents** | **Apparatus** |
| Vector (digested pCMV-3Tag-1A) | Micro-centrifuge tube (1.5 mL) |
| OGN fragment | Pipette (range: 1 μL-10 μL) |
| T4 DNA Ligase | Pipette (range: 0.1 μL-2.5 μL) |
| 10× T4 DNA Ligase Buffer | Centrifuge |
| ddH2O | Thermostat |

2) Procedure

Ø Prepare reaction solution in a tube

|  |  |
| --- | --- |
| **Reagents** | **Volume/mass** |
| Vector (digested pCMV-3Tag-1A) | 0.03 pmol |
| OGN fragment | 0.3 pmol |
| T4 DNA Ligase | 1 μL |
| 10× T4 DNA Ligase Buffer | 1 μL |
| ddH2O | to 10 μL |

Ø Centrifuge the liquid to the bottom of the tube.

Ø Place the tube in the thermostat at 16℃ overnight.

E. coli transformation

1) Materials

|  |  |
| --- | --- |
| **Reagents** | **Apparatus** |
| pCMV-3Tag-1A-OGN | Pipette (range: 1 μL-10 μL) |
| DH5α chemically competent cell | Pipette (range: 1 μL-100 μL) |
| LB liquid medium (Kan-) | Petri dish |
| LB solid medium (Kan+) | Thermostat |
|  | Shaker |
|  | Incubator |

2) Procedure

Ø Transformation

u Thaw 30 μL of competent cells on ice for 1-2 min.

u Mix the cells with 1 μL of plasmid (pCMV-3Tag-1A-OGN) in a
tube.

u Place the tube on ice for 30 min.

u Heat shock: place the tube in the thermostat at 42℃ for 45
s.

u Place the tube in ice bath immediately and cool it for 2-3
min.

Ø Resuscitation

u Add 1 mL of LB liquid medium (Kan-) to the tube.

u Mix the solution uniformly, and incubate it in the shaker at
37℃, 220 rpm for 45-60 min.

Ø Incubation

u Aspirate 100 μL of transformed competent cells with one
pipette.

u Coat them uniformly on the LB solid media (Kan+).

u Place the petri dish at room temperature until the solution is
absorbed.

u Turn the dish upside down and incubate the cells at 37℃ for
12-16 h.

Plasmid extraction

1) Materials

|  |  |
| --- | --- |
| **Reagents** | **Apparatus** |
| TIANpure Midi Plasmid Kit | Micro-centrifuge tube (1.5 and 2 mL) |
| Bacterial solution | Pipette (range: 0.1 mL-1 mL) |
| PBS | Vortex machine |
|  | Centrifuge |

2) Procedure

Ø Follow the manufacturer’s instruction.

Agarose gel electrophoresis

1) Materials

|  |  |
| --- | --- |
| **Reagents** | **Apparatus** |
| Agarose | Conical flask (range: 250 mL) |
| TAE buffer | Pipette (range: 1 μL-10 μL) |
| YeaRed Nucleic Acid Gel Stain (10000×) | Electronic balance |
| DNA loading buffer (10×) | Spatula |
| DNA Marker | Microwave oven |
| DNA sample | Mold for creating gel |
|  | Electrophoresis chamber |
|  | UV transilluminator |
|  | Scalpel |

2) Procedure

Ø Gel preparation

u Place the weighing paper on the electronic balance, weigh 1g of
agarose, and pour it into the conical flask.

u Pour 100 mL of TAE buffer into the conical flask and shake
it.

u Cover the conical flask with the aluminum foil and place it
into the microwave oven for 3 min, until the granules of agarose
are well dissolved and invisible.

u Use the pipette to transfer 10 μL of YeaRed nucleic acid gel
stain into the conical flask and shake it.

u Pour the gel into the mould and insert a “comb”
into the gel.

u Wait for 10 min until the gel is solidified.

Ø Electrophoresis

u Pour about 400 mL of TAE buffer into the electrophoresis
chamber.

u Carefully take out the gel and place it in the electrophoresis
chamber.

u Mix the loading buffer and DNA sample by using the
pipette.

u Use the pipette to add 5 μL of marker in the first hole of
the gel, then add 10 μL of each sample into the following
holes.

u Turn on the power of the electrophoresis chamber and wait for
about 30 min (the specific time should depend on the loading
buffer we used).

Ø Authentication and recycle

u Take out the gel and place it on the UV tray. The UV tray will
then be placed into the UV transilluminator.

u Scan the gel.

u Comparing with the marker reference, determine the target bands
which will be extracted during the recycling process.

u Use the scalpel to cut the target bands of the gel.

![](https://static.igem.wiki/teams/4840/wiki/ex1.png)

DNA Gel Extraction

1) Materials

|  |  |
| --- | --- |
| **Reagents** | **Apparatus** |
| TIANgel Midi Purification Kit | Micro-centrifuge tube (1.5 mL) |
| Agarose gel | Pipette (range: 0.1 mL-1 mL) |
|  | Centrifuge |

2) Procedure

Ø Follow the manufacturer’s instruction.

ATDC5 cells transformation

1) Materials

|  |  |
| --- | --- |
| **Reagents** | **Apparatus** |
| pCMV-3Tag-1A-OGN | Tube (1.5 mL) |
| ATDC5 cells | Pipette (range: 1 μL-10 μL) |
| Lipofectamine 2000 | Pipette (range: 1 μL-100 μL) |
| MEM medium | Pipette (range: 0.1 mL-1 mL) |
| OPTI-MEM medium | Incubator |

2) Procedure

Ø Add 2 μg of pCMV-3Tag-1A-OGN, 2 μL of Lipofectamine 2000
into a tube with 200 μL of OPTI-MEM medium.

Ø Mix the solution gently and incubate at room temperature for 20
min.

Ø Replace the cell growth medium with 800 μL of MEM
medium.

Ø Add the solution to the cell culture plate and shake
gently.

Ø Incubate the cells at 37℃ for 4 h.

Ø Replace the MEM medium with the cell growth medium.

Ø Incubate the cells at 37℃ for another 24 h.

Alcian blue dyeing

1) Materials

|  |  |
| --- | --- |
| **Reagents** | **Apparatus** |
| 4% paraformaldehyde | Centrifuge tube (range: 15 mL) |
| Alcian blue 8GX | Electronic balance |
| Acetic acid | Pipette (range: 0.1 mL-1 mL) |
| PBS | Shaker |
| dH2O | Microscope |

2) Procedure

Ø Prepare 1% Alcian blue dyeing solution.

|  |  |
| --- | --- |
| **Reagents** | **Volume/mass** |
| Alcian blue 8GX | 1 g |
| Acetic acid | 3 mL |
| dH2O | 97 mL |

Ø Remove the cell growth medium and wash the cells with
PBS.

Ø Fix the cells with 4% paraformaldehyde at room temperature for
15 min.

Ø Wash the cells with dH2O for three times (5 min each
time).

Ø Incubate the cells with Alcian blue dyeing solution at 4℃
overnight.

Ø Recycle the dye solution.

Ø Wash the cells with dH2O for three times (5 min each
time).

Ø The cells were maintained in dH2O and captured by using a
microscope.

SDS-PAGE electrophoresis

1) Materials

|  |  |
| --- | --- |
| **Reagents** | **Apparatus** |
| Protein sample | Pipette (range: 1 μL-10 μL) |
| Running buffer | Pipette (range: 0.1 μL-2.5 μL) |
| Loading buffer | Electrophoresis chamber |
| Marker |  |
| PAGE (12%) |  |

2) Procedure

Ø Insert the prepared gel (12% BT) into the electrophoresis
chamber.

Ø Add running buffer into the chamber until the gel is
submerged.

Ø Mix the protein sample and loading buffer.

Ø Add 1.5 μL of marker to the first hole of the gel and add 10
μL of mixed sample solution to the following holes.

Ø Set up the electrophoresis program.

|  |  |  |
| --- | --- | --- |
| Step 1 | 80 V | 30 min |
| Step 2 | 120 V | 60 min |

Ø Start the program.

Western blot

1) Materials

|  |  |
| --- | --- |
| **Reagents** | **Apparatus** |
| Rabbit anti-OGN antibody | Trans-blot transfer |
| Mouse anti-β-actin antibody | NC membrane |
| HRP-conjugated Goat Anti-Rabbit IgG | Shaker |
| HRP-conjugated Goat Anti-Mouse IgG | Box (1 dm×1 dm×0.5 dm) |
| TBST solution |  |
| Clarity Western ECL Substrate |  |
| 5% defatted milk solution |  |
| Gel with protein after SDS-PAGE |  |

2) Procedure

Ø Transfer

u Place the gel and NC membrane in the trans-blot
transfer.

u Submerge trans-blot transfer in the ice bath.

u Set the transfer program at 320 mA, 90 min.

u Start the program.

Ø Blocking

u Turn the membrane to a box, add TBST solution into it, and
rinse the membrane for 1-2 min.

u Discard the TBST solution.

u Add 5% defatted milk into the box until the membrane is
submerged.

u Place the box on the shaker at room temperature for 2 h.

Ø Primary antibody incubation

u Discard the milk in the box.

u Add rabbit anti-OGN antibody or mouse anti-β-actin
antibody solution to the box.

u Place the box on the shaker at 4℃ overnight.

u Recycle the primary antibody solution.

u Rinse the membrane with TBST solution three times, 15 min for
each time.

Ø Secondary antibody incubation

u Discard the TBST solution in the box.

u Add HRP-conjugated Goat Anti-Rabbit IgG or HRP-conjugated Goat
Anti-Mouse IgG solution to the box.

u Place the box on the shaker at room temperature for 2 h.

u Recycle the secondary antibody solution.

u Rinse the membrane with TBST solution three times, 15 min for
each time.

Ø Detection of proteins

u Add Clarity Western ECL Substrate solution into the box until
the membrane is evenly soaked.

u Use the software GelCap to scan the membrane.

![](https://static.igem.wiki/teams/4840/wiki/ex2.png)

Quantitative Real-Time PCR (qRT-PCR)

1) Materials

|  |  |
| --- | --- |
| **Reagents** | **Apparatus** |
| ChamQ SYBR qPCR Master Mix (2×) | PCR tubes (0.1 mL) |
| Template cDNA | Pipette (range: 1 μL-10 μL) |
| Forward primer (20 μM) \* | Pipette (range: 0.1 μL-2.5 μL) |
| Reverse primer (20 μM) \* | qPCR thermal cycler |
| ddH2O |  |

2) Procedure

Ø Prepare reaction solution.

|  |  |
| --- | --- |
| **Reagents** | **Volume** |
| ChamQ SYBR qPCR Master Mix (2×) | 10 μL |
| Template cDNA | 0.1 μL |
| Forward primer (20 μM) \* | 0.25 μL |
| Reverse primer (20 μM) \* | 0.25 μL |
| ddH2O | 9.5 μL |

Ø Set up the reaction program.

|  |  |  |
| --- | --- | --- |
| Step 1 | 95℃ | 10 s |
| Step 2 | 95℃ | 5 s |
| Step 3 | 56℃ | 20 s |
| Step 4 | 72℃ | 10 s |
| Step 5 | Goto step 2, 45 more times | |
| Step 6 | 72℃ | 10 min |
| Step 7 | Melt Curve 50 to 95℃, increment 0.5℃ for 5 s | |
| Step 8 | 10℃ | 5 min |
| End | | |

Ø Start the program.

\* Sequence of primers：

|  |  |  |
| --- | --- | --- |
| **Gene** | **Forward primer** | **Reverse primer** |
| OGN | ACCATAACGACCTGGAATCTGT | AACGAGTGTCATTAGCCTTGC |
| Col2α1 | GGGAATGTCCTCTGCGATGAC | GAAGGGGATCTCGGGGTTG |
| Aggrecan | CCTGCTACTTCATCGACCCC | AGATGCTGTTGACTCGAACCT |
| Sox9 | GAGCCGGATCTGAAGAGGGA | GCTTGACGTGTGGCTTGTTC |
| MMP3 | ACATGGAGACTTTGTCCCTTTTG | TTGGCTGAGTGGTAGAGTCCC |
| MMP13 | CTTCTTCTTGTTGAGCTGGACTC | CTGTGGAGGTCACTGTAGACT |
| β-Actin | GGCTGTATTCCCCTCCATCG | CCAGTTGGTAACAATGCCATGT |

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![](https://static.igem.wiki/teams/4840/wiki/results.png)

Overview

With the aging of the population and the increase of obese people, the prevalence of OA is rising. OA is a
degenerative disease characterized by cartilage destruction and loss, subchondral bone thickening and
sclerosis, osteophyte formation with varying degrees of synovitis and joint capsule thickening[1][ Choi WS,
Lee G, Song WH, Koh JT, Yang J, et al. The CH25H-CYP7B1-RORalpha axis of cholesterol metabolism regulates
osteoarthritis. Nature. 2019 Feb;566(7743):254-58.]. Recent studies have found that the expression of OGN in
articular cartilage tissues of OA patients is significantly reduced, and OGN is involved in the regulation
of matrix metabolism in various tissues[2][ Nulali J, Zhan M, Zhang K, et al. Osteoglycin: An ECM Factor
Regulating Fibrosis and Tumorigenesis. Biomolecules. 2022 Nov 11;12(11):1674.]. However, its regulatory role
in matrix metabolism of OA chondrocytes needs further exploration. In this study, we will construct a cell
model of chondrocyte matrix metabolism disorder induced by inflammatory factor IL-1β, and use the
intervention method of gene overexpression to clarify the regulation of OGN expression in chondrocytes by
inflammatory factors(Figure 1). To determine the effect of OGN on IL-1β-regulated chondrocyte matrix
metabolism.

![](https://static.igem.wiki/teams/4840/wiki/result/1.png)

I.pCMV-3Tag-1A-OGN

1)Construction of OGN overexpressed plasmid

Choi WS, Lee G, Song WH, Koh JT, Yang J, et al. The CH25H-CYP7B1-RORalpha axis of cholesterol metabolism
regulates osteoarthritis. Nature. 2019 Feb;566(7743):254-58.

Nulali J, Zhan M, Zhang K, et al. Osteoglycin: An ECM Factor Regulating Fibrosis and Tumorigenesis.
Biomolecules. 2022 Nov 11;12(11):1674.

In this work, we first constructed OGN overexpressed plasmid. In brief, DNA fragment of OGN was amplified
by using the cDNA of ATDC5 cells and then inserted into the pCMV-3Tag-1A vector. The E. coli strains
solution transformed with constructed plasmid were spread onto LB solid medium plate containing
corresponding antibiotic and cultured at 37℃ overnight (Figure 2).

![](https://static.igem.wiki/teams/4840/wiki/result/2.png)

Figure 2. Monoclonal colonies of E. coli transformed with pCMV-3Tag-1A-OGN plasmid

We selected several monoclonal colonies from the plate for cultivation, and extracted plasmid DNA from the
cultured bacterial solution. The oligo DNA in plasmid was identified by using agarose gel electrophoresis
(Figure 3A). Our results show that a band of about 5000 bp is present in sample 2 and 8, suggested that
pCMV-3Tag-1A-OGN plasmid were constructed successfully. At the same time, we sent the positive monoclonal
antibody to the company for sequencing. Figure 3B showed that the gene sequence was correct and there was no
mutation. It proves that pCMV-3Tag-1A-OGN plasmid was successfully constructed.

![](https://static.igem.wiki/teams/4840/wiki/result/3.png)

Figure 3. Electrophoretic gel (left) and Sequencing (right) Verification of pCMV-3Tag-1A-OGN plasmid

2)Identification of ITS-induced ATDC5 cells

In order to explore the function of OGN on the matrix metabolism of chondrocytes. We firstly added ITS to
the growth medium to induce the differentiation of ATDC5 cells. Since mature chondrocytes contain a large
amount of proteoglycan, which can be stained blue by Alcian Blue, we performed related assays in ITS-induced
ATDC5 cells. As shown in Figure 4, our cultured ATDC5 cells have differentiated into chondrocytes.

![](https://static.igem.wiki/teams/4840/wiki/result/4.png)

Figure 4. Microscope image of chondrocytes

3)The increase of OGN expression improved matrix metabolism in IL-1β-treated chondrocytes

Next, we established an in vitro research model of OA by using IL-1β as stimulator on chondrocytes and
detected the mRNA and protein levels of OGN in the cells by using qRT-PCR and western blotting. As shown in
the Figure 5, the mRNA and protein levels of OGN in IL-1β-stimulated chondrocytes were significantly
decreased compared with those of control group, that is consistent with the clinical observation that OGN
expression in chondrocytes from osteoarthritis patients is reduced. Moreover, both the mRNA and protein
levels of OGN in chondrocytes transfected with previously constructed pCMV-3Tag-1A-OGN plasmid were notably
increased compared to the mock group, further indicating the overexpressed plasmid was successfully
constructed and worked successfully.

![](https://static.igem.wiki/teams/4840/wiki/result/5.png)

Figure 5. pCMV-3Tag-1A-OGN plasmid antagonisted IL-1β-induced inhibition on OGN expression in chondrocytes
(A) qRT-PCR results; (B) western blot results

Additionally, we investigated whether OGN participates in the pathogenesis of OA. As shown in Figure 6,
mRNA levels of matrix metabolism-related genes such as Aggrecan, Col-2 and Sox9 were downregulated, while
MMP3 and MMP13 mRNA levels were upregulated in IL-1β-induced chondrocytes. These results indicated the
disruption of matrix metabolism in chondrocytes. Notably, OGN overexpression antagonized such IL-1β-induced
effects in chondrocytes. Thus, all these results suggests that OGN is a promising therapeutic target for OA.

![](https://static.igem.wiki/teams/4840/wiki/result/6.png)

Figure 6. OGN overexpression antagonisted IL-1β-induced matrix metabolism disorder in chondrocytes

II.RNAi-ReadypSIREN-RetroQ-ShRNA-1;RNAi-ReadypSIREN-RetroQ-ShRNA-2; RNAi-Ready pSIREN-RetroQ-ShRNA-N

1)Construction of OGN gene silencing plasmids

Moreover, we designed and synthesized two interference sequences of OGN gene for the construction of OGN
gene silencing plasmids and performed similar experiments to explore whether the OGN is involved in the
initiation and progression of OA.

Briefly, the forward and reverse sequences of ShRNA were connected by annealing. The obtained DNA fragments
was inserted into the RNAi-Ready pSIREN-RetroQ vector. The E. coli strains solution transformed with
constructed plasmid were spread onto LB solid medium plate containing corresponding antibiotic and cultured
at 37℃ overnight (Figure 7).

![](https://static.igem.wiki/teams/4840/wiki/result/7.png)

Figure 7. Monoclonal colonies of E. coli transformed with OGN gene silencing plasmids (A) RNAi-Ready
pSIREN-RetroQ-ShRNA-1; (B) RNAi-Ready pSIREN-RetroQ-ShRNA-2; (C) RNAi-Ready pSIREN-RetroQ-ShRNA-N

We selected several monoclonal colonies from the plate for cultivation, and extracted plasmid DNA from the
cultured bacterial solution. The oligo DNA in plasmid was identified by using agarose gel electrophoresis
(Figure 8A). At the same time, we sent the positive monoclonal antibody to the company for sequencing.
Figure 8 B. C.D showed that the gene sequence was correct and there was no mutation.

It proves that RNAi-Ready pSIREN-RetroQ-ShRNA-1, RNAi-Ready pSIREN-RetroQ-ShRNA-2, RNAi-Ready
pSIREN-RetroQ-ShRNA-N plasmid were successfully constructed.

![](https://static.igem.wiki/teams/4840/wiki/result/8.png)

Figure 8. Electrophoretic gel (A) and Sequencing (B.C.D) Verification of RNAi-Ready pSIREN-RetroQ plasmid

4)OGN gene silencing can induce matrix metabolism disorder in chondrocytes

As shown in Figure 9 and Figure 10, the mRNA and protein levels of OGN were significantly decreased in
IL-1βgroup and OGN-Sh-1, 2 group compared with those of Control group or OGN-Sh-N group. In addition, the
results of qRT-PCR revealed that the decrease of OGN expression can induce the disruption of matrix
metabolism in chondrocytes, indicative of the regulatory role of OGN in OA.

![](https://static.igem.wiki/teams/4840/wiki/result/9.png)

Figure 9. RNAi-Ready pSIREN-RetroQ-ShRNA-1 and 2 plasmids inhibited the expression of
OGN in chondrocytes (A) qRT-PCR results; (B) western blot resultsFigure 8. Electrophoretic gel (A) and
Sequencing (B.C.D) Verification of RNAi-Ready pSIREN-RetroQ plasmid

![](https://static.igem.wiki/teams/4840/wiki/result/10.png)

Figure 10. The decrease of OGN expression induced matrix metabolism disorder in
chondrocytes

References and sources

Choi WS, Lee G, Song WH, Koh JT, Yang J, et al. The CH25H-CYP7B1-RORalpha axis of cholesterol
metabolism regulates osteoarthritis. Nature. 2019 Feb;566(7743):254-58.

Nulali J, Zhan M, Zhang K, et al. Osteoglycin: An ECM Factor Regulating Fibrosis and Tumorigenesis.
Biomolecules. 2022 Nov 11;12(11):1674.

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