Hippo Pathway trans-Reporting System

$828.00Catalog Number: BXH1001-hipSize: kit

transLight^TM Hippo Pathway Luciferase Reporting system

Hippo Pathway trans-reporting system is designed for specific, rapid in vivo read-out of the activation of transcription factor TEAD4 and its upstream Hippo signaling pathway, which plays key roles in cell growth, proliferation, apoptosis and organ size control.

The system uses Gal4-TEAD4 fusion protein, which is expressed from the Activator Plasmid pGAL4-TEAD, as the sensor of the activation of Hippo kinase cascade. The transcription coactivator YAP works together with Gal4-TEAD4 to activate luciferase expression from pHTS-GAL4 vector when transfected into mammalian cells.

Activated Hippo kinase cascade in the cell results in the phosphorylation of YAP and its translocation to the cytoplasm. Less YAP in the nucleus, in turn, causes reduced luciferase expression. Inhibition of Hippo kinase cascade, however, results in elevated luciferase activity. This system is useful to asses whether a gene is involved or affecting the various components along the Hippo pathway. It can also be used to study the in vivo effects of growth factors, drug candidates, and extracellular stimuli.

Includes and/or available separately

Plasmid Name	pHTS-GAL4	pGAL4-TEAD4	pGAL4-DBD	pM2H-YAP2A5
Plasmid Function	Reporter	Activator	Negtive Control	Positive Control
Selection Markers	Amp/Hygro	Kan/G418	Kan/G418	Kan/G418
Gene of Interest	Firefly Luciferase	GAL4 and TEAD4	DNA binding domain	Activated YAP2
Promoter:	5xGBE	CMV early	CMV early	CMV early
Quantity	40 ug/80 ul	2 ug/80 ul	2 ug/80 ul	2 ug/80 ul
Part Number	BXH1001-rep	BXH1001-act	BXH1001-neg	BXH1001-pos

Individual plasmids and custom sizes are available upon request.

The Activator Plasmid

The fusion trans-activator plasmid pGAL4-TEAD4 contains the human cytomegalovirus (CMV) immediate early promoter to drive the constitutive expression of the trans-activator protein in a wide variety of eukaryotic cell lines.

The fusion trans-activator GAL4-TEAD4 consists of the DNA binding domain (DBD) of yeast GAL4 (residues 1-147) and the activation domain of human TEAD4 (NM_003213, residues M74-E434). The GAL4 DBD domain allows the specific binding of the fusion protein to the GAL4 binding elements (GBE) in the Reporter Plasmid (pHTS-GAL4); The TEAD4 activation domain, when activated by upstream YAP2, activates luciferase gene expression from pHTS-GAL4 plasmid.

The activator plasmids can be selected with Kanamycin that is conferred by the kanamycin-resistance gene under control of the prokaryotic &betta;-lactamase promoter. The neomycin-resistance gene, driven by the SV40 early promoter, confers stable selection with G418 in mammalian cells.

Reporter Plasmid

The reporter plasmid pHTS-GAL4 contains an expression cassette for the firefly luciferase under the control of GAL4-binding element (CGGAGTACTGTCCTCCG AG)x5. Luciferase expression is activated in mammalian cells when a trans-activator (e.g. GAL4-TEAD) binds to the GBE.

Control Plasmids

The negative control plasmid pGAL4-DBD expresses the GAL4 DNA binding domain (aa 1-147), which can bind to GBE in pHTS-GAL4 plasmid but is incapable of activation luciferase expression. The positive control plasmid pM2H-YAP2A5 is a constitutively active human YAP2 mutant that activates TEAD4 independent of activation of upstream components in the Hippo signaling pathway.

Transfection and cell selection

Plasmids provided in the kit are highly purified and ready for transfection with any standard transfection reagenents and protocols such as liposome reagents, electroporation or Calcium Phosphate Precipitation. Refer to the manufacturers' instruction manuals for detailed protocols.

Different types of mammalian cells vary widely in endogenous signaling proteins and other properties, leading to different sensitivities of the assay. Assay conditions should be optimized empirically.

Sample experiment design

Experiments should be designed with the proper controls according to the purpose of the study. The examples given below can serve as a starting point. The exact conditions, especially the amount of positive control plasmid, the experimental plasmid and extracellular stimuli should be determined experimentally. The examples given are based on 24-well tissue culture plate. If plates of different sizes are being used, the amount of all reagents should be adjusted according to the area of the wells or dishes. Ideally, each sample should be run in triplicate.

Prepare plasmid mixtures as listed below for transfection (unit: ng):

Sample Number	1	2	3	4	5	6	7	8
pHTS-GAL4	400	400	400	400	400	400	400	400
pGAL4-TEAD4	25	25	25	25	25	25	25	-
Target Gene(e.g. pM2H-Mst2)	25	50	100	-	-	-	-	50
Control (e.g. pM2H vector)	-	-	-	25	50	100	-	-
Positive control	-	-	-	-	-	-	25	-
pGAL4-DBD	-	-	-	-	-	-	-	25

Note: 1) The plasmid used in #4-6 is ideally the parental plasmid used for expression of the gene of interest. 2) Sample #7 is the positive control that assesses the efficacy of the assay. 3)pGAL4-DBD in sample #8 does not express GAL4-TEAD4 and is a negative control. 4) Ideally, a carrier DNA (e.g. pUC18) should be used to make all the samples have the same amount of DNA. 5) The exact amount (or the range) of the experimental plasmid should be determined experimentally.

Luciferase Activity Assay

Use of a commercial luciferase assay may be more convenient. The following protocol is provided for quick reference only.

Remove media from cell and rinse twice with PBS and remove residual PBS
Add 1x Lysis Buffer (e.g. 400ul per well of a six-well plate, see below for buffer components). Incubate the plate for 15 minutes at room temperature (RT) with gentle rocketing.
The lysates could be used in luciferase assay or be transferred to microcentrifuge tubes and stored at -80^oC
Mix 5-20ul of cell lysate with 100ul of 1X Luciferase Assay Buffer in an appropriate tube (e.g. Falcon 2054 polystyrene tube). All reagents should be brought to RT for assay.
Measure the light emission with a luminometer with an integration time of 10-30 seconds.

5X Lysis Buffer: Tricine (pH7.8) 40mM; NaCl 50mM; EDTA 2mM; MgSO₄ 1mM; DTT 5mM; Triton X-100 1%.
Assay Buffer (1x): Tricine (pH7.8) 40mM; ATP 0.5mM; MgSO₄ 10mM; EDTA 0.5mM; DTT 10mM; Coenzyme A 0.5mM and luciferin 0.5mM.

Select references

Zhao B. et al., 2007. Genes Dev., 21: 2747-61
Zhao B. et al., 2008. Genes Dev., 22: 1962-1971
Dong J. et al., 2007, Cell, 130: 1120-1133
Zhang L. et al., Dev Cell, Epub March 2008, doi:10.1016/j.devce.2008.01.006
Sadowski, I. and Ptashne, M. (1989) Nucleic Acids Res 17(18):7539
Wu S. et al., Dev Cell, Epub March 2008, doi:10.1016/j.devce.2008.01.006