Difference between revisions of "Part:BBa K1699001"

Revision as of 09:28, 13 September 2015

Human short TERT promoter

Human short TERT promoter.

hTERT (human Telomerase Reverse Transcriptase) is a human promoter which controls the transcription of Telomerase, a gene highly expressed in cancer cells, and not in healthy ones (1).

Usage and Biology

hTERT promotes the transcription of the catalytic subunit of telomerase. Telomerase elongates the ends of chromosomes, regions called telomers. hTERT is not active in somatic cells and is highly active in most if not all cancer cell types. Therefore, the expression of an exogenous gene under the control of hTERT promoter will likely occur in cancer cells only. We have used this promoter as part of a two promoter system (the other one being survivin promoter, also highly active in cancer cells), in order drive the expression, and subsequently activate the core of our system (based on CRISPR?Cas9 technology) exclusively in cancer cells.

Characterization

The validity of hTERT promoter for the application of cancer-specific gene expression was performed using quantitative real-time PCR (qPCR), where the expression of hTERT was evaluated in several human cancer cell lines comprared to healthy cells (fibroblasts) (Fig. 1). The results show thousand-fold and higher TERT expression levels in cancer cells, suggesting marked promoter hyperactivation.

Fig. 1. hTERT epxression level in several human cancer cell types, evaluated by qPCR. HF-human fibroblasts (healthy cells); cancer cell lines: HepG2 - hepatocarcinoma, A549 - lung adenocarcinoma, MDA-MB-231 - breast adenocarcinoma, HT1080 - fibrosarcoma.

This part was used and validated by BGU 2015 team in a following constructs.
1. phTERT-GFP: AAV (adeno-acociated virus)- vector expressing GFP under the control of human short TERT promoter was constructed (Fig. 2).

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Fig. 2. Plasmid map of AAV vector expressing eGFP under the control of human short TERT promoter.

Following calcium phoshpate plasnid transfection or AAV transduction, robust GFP expression was evident only in cancer cells, compared to undetected levels in healthy cells (Fig. 3).

Fig. 3. eGFP expression under short human TERT promoter in cancer cells (fibrosarcoma) and healthy fibroblasts, after caclium phosphate plasmid transfection (A) or AAV transduction (B). Bar: 200 micron.

2. phTERT-dCas9-VP64: AAV vector expressing dCas9-VP64 (2) (engineered version of "classical" Cas9 for transcriptional activation of any gene of interest) under the control of human short TERT promoter was constructed as a part of functional prototype of two cancer-specific promoter-driven CRISPR/Cas9 activation system of BGU 2015 Boomerang team (Fig. 4).

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Fig. 4. Plasmid map of AAV vector expressing dCas9-VP64 under the control of human short TERT promoter.

Following simultaneous plasmid transfection of dCas9-VP64 - under the control of hTERT promoter, guide RNA (targeting the synthetic activation promoter) - under the control of human survivin promoter, and eGFP under synthetic activation promoter (3), eGFP expression was detected only in cancer cells, compared to undetected levels in healthy cells (Fig. 5). The unique ribozyme design was used to drive gRNA synthesis under "unusual" control of RNA polymerase II (and not III) promoter (4, 5).

Fig. 5. eGFP expression from synthetic activation promoter exclusively in cancer cells after successful activation of CRISPR-based activation core driven by dCas9-VP64 - under the control of hTERT promoter, and guide RNA (targeting the synthetic activaiton promoter) - under the control of human survivin promoter. Bar:100 micron.

The expression of dCas9-VP64 and gRNA under the control of cancer-specific promoters (TERT and survivin) drives the activation of the system only in cancer cells (Fig. 6).

Fig. 6. Cancer-specific CRISPR-based activation of the gene of interest using two cancer-specific promoter-driven expression of dCas9-VP64 and gRNA.

3. phTERT-SaCas9: We also engineered AAV construct in which the short human TERT controls the expression of "classical" Cas9 (SaCas9 (6)) (Fig. 7A). The construct, together with gRNA under the control of human survivin promoter link to registry part, could drive cancer-specific gene knockout (Fig. 7B).

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Fig. 7. A. Plasmid map of AAV vector expressing SaCas9 under the control of human short TERT promoter. B. Summary of two cancer-specific promoter-driven CRISPR-mediated gene knock-out.

References

1. The telomerase reverse transcriptase promoter drives efficacious tumor suicide gene therapy while preventing hepatotoxicity encountered with constitutive promoters http://www.nature.com/gt/journal/v8/n7/pdf/3301421a.pdf

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 41: / Line 41: @@
 [[Image:PhTERT-dCas9-VP64-polyA-pAAV_Map.jpg|center|500px|thumb|'''Fig. 4'''. Plasmid map of AAV vector expressing dCas9-VP64 under the control of human short TERT promoter.]]
-Following simultaneous plasmid transfection of dCas9-VP64 - under the control of hTERT promoter, guide RNA (targeting the synthetic activation promoter) - under the control of human survivin promoter, and eGFP under synthetic activation promoter, eGFP expression was detected only in cancer cells, compared to undetected levels in healthy cells (Fig. 5). The unique ribozyme design was used to drive gRNA synthesis under "unusual" control of RNA polymerase II (and not III) promoter (3, 4).
+Following simultaneous plasmid transfection of dCas9-VP64 - under the control of hTERT promoter, guide RNA (targeting the synthetic activation promoter) - under the control of human survivin promoter, and eGFP under synthetic activation promoter (3), eGFP expression was detected only in cancer cells, compared to undetected levels in healthy cells (Fig. 5). The unique ribozyme design was used to drive gRNA synthesis under "unusual" control of RNA polymerase II (and not III) promoter (4, 5).
 [[Image:Activation.jpg|center|500px|thumb|'''Fig. 5'''. eGFP expression from synthetic activation promoter exclusively in cancer cells after successful activation of CRISPR-based activation core driven by dCas9-VP64 - under the control of hTERT promoter, and guide RNA (targeting the synthetic activaiton promoter) - under the control of human survivin promoter. Bar:100 micron.]]
@@ Line 48: / Line 48: @@
 [[Image:Activ2(full).jpg|center|500px|thumb|'''Fig. 6'''. Cancer-specific CRISPR-based activation of the gene of interest using two cancer-specific promoter-driven expression of dCas9-VP64 and gRNA.]]
+<br />3. '''phTERT-SaCas9''': We also engineered AAV construct in which the short human TERT controls the expression of "classical" Cas9 (SaCas9 (6)) (Fig. 7A). The construct, together with gRNA under the control of human survivin promoter [https://parts.igem.org/Part:BBa_K1699002 link to registry part], could drive cancer-specific gene knockout (Fig. 7B).
+[[Image:PhTERT-SaCas9-polyA-pAAV_Map.jpg|center|500px|thumb]]
+[[Image:Knock-out_(full).jpg|center|500px|thumb|'''Fig. 7'''. A. Plasmid map of AAV vector expressing SaCas9 under the control of human short TERT promoter. B. Summary of two cancer-specific promoter-driven CRISPR-mediated gene knock-out.]]
 ===References===