Part:BBa_K1722000
hUPll | |
---|---|
Function | Promoter |
Use in | Bladder cells |
RFC standard | RFC 10 |
Backbone | pSB1C3 |
Submitted by | [http://2015.igem.org/Team:SZU_China SZU_China 2015] |
hUPll is a bladder tissue-specific promoter .
hUPll is a bladder tissue-specific promoter being found in human urothelium. Uroplakin II (UPII) has been characterized as a bladder tissue-specific protein[1] and the expression of uroplakin II was found to be limited to bladder-derived cells.[2,3] Other members of uroplakins, including uroplakinla(UPla), uroplakinlb(UPlb), and uroplakinlll(UPlll), have also been characterized. Therefore, the promoters that direct the expression of the uroplakins may be useful in constructing tissue-specific vectors for bladder cancer gene therapy. Research shows that most of the elements that confer the bladder-specificity and differentiation-dependent expression of the human UPll gene reside in the 2542-bp sequence, and TNF driven by the human UPll(hUPll) promoter is effective in the specific inhibition of bladder cancer growth both in vivo and in vitro. Zhu et al transtected the plasmid phUPll-EGFP containing DNA fragment(hUPll) into bladder cancer(BIU-87), renal carcinoma(GRC-1) and endothelial(EC) cell lines.[4](Fig. 1)
hUPll is 355bp in length. Fig. 2 shows the DNA sequence of hUPll is successfully amplified by PCR from psi-Check2 vector. From this electrophoretogram, we can see the brightness of hUPll PCR product is rather high compared with DNA Marker, which indicates that the PCR product of hUPll is in a high concerntration.
After ligating hUPll and pSB1C3, we transfected the new pasmid being constructed into Ecoli and selected those Ecoli with Chl resistence. Using these Ecoli as templet, we amplified hUPll from pSB1C3,(Fig. 3) which means we had successfly construacted the pSB1C3-hUPll plasmid.
We then performed single digest(EcoRI) and double digest(EcoRI & PstI) to identify our pSB1C3-hUPll plasmid. From the eletrophoretogram, we have an electrophoresis strip at about 2425bp in Track 1 and two strips at about 355bp, which is exactly the length of hUPll promoter and 2070bp, which is the length of pSB1C3 in Track 2. From this enzyme cutting result, we could make sure the Gene sequence of hUPll succeeded in being constructed into pSB1C3 vector.
2015 SZU-iGEM use hUPII to drive the expression of the therapeutic genes(such as p21 and Bax) so that the gene is expressed only in bladder cells and systematic toxicity is minimized. We tested hUPII promoter in HFC(Human Fiber Epithelial Cells),5637,T24 and Hela cell lines. HFC are normal bladder cells, 5637 and T24 are bladder cancer cells while Hela are cervical cancer cells. Result shows it can confer preferential expression of genes in the bladder urothelium like HFC, 5637 and T24, which indicates hUPll has high efficiency in specifically recognizing bladder cells.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
We designed the following primers and amplified hUPII promoter from the vector psi-Check2:
CCGGAATTCATCGGGTGATCAGTACTCC(up) TGCACTGCAGACTAGTACTGAGCTGTGAGGT(down)
By incorporating these primers into hUPII promoter, the promoter is flanked by the iGEM prefix and suffix after amplification.
Source
hUPII gene was achieved from Shenzhen Second People's Hospital. We read a scientific treatise talking about targeted therapy of bladder cancer written by a doctor in Shenzhen Second People's Hospital and tried to seek cooperation with them. Fortunately, they agree to provide us hUPII with psi-Check2 as its vector.
References
[1]Wu XR, Lin JH, Walz T, et al. Mammalian uroplakins, a group of highly conserved urothelial differentiation related membrane proteins. J Biol Chem. 1994;269:13716–13724.
[2]Yuasa T, Yoshiki T, Isono T, et al. Expression of transitional cell specific genes uroplakin Ia and II in bladder cancer detection of circulating cancer cells in the peripheral blood of metastatic patients. Int J Urol. 1999;6:286–292.
[3]Moll R, Wu XR, Lin JH, Sun TT. Uroplakins specific membrane proteins of urothelial umbrella cells as histological markers of metastatic transitional cell carcinomas. Am J Pathol. 1995;147:1383–1397.
[4]Zhu H J, Zhang ZQ, Zeng XF, et al. Cloning and analysis of human uroplakin II promoter and its ap plication for gene therapy in bladder cancer[J] Cancer Gene Ther, 2004, 11: 263-272
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