STOP. Before using this product, please read the Limited Use License statement below:

Transcription

1 STOP Before using this product, please read the Limited Use License statement below: Important Limited Use License information for pdrive5lucia-hnphsi The purchase of the pdrive5lucia-hnphsi vector conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. The buyer may transfer information or materials made through the use of this product to a scientific collaborator, provided that such transfer is not for any Commercial Purpose, and that such collaborator agrees in writing (a) not to transfer such materials to any third party, and (b) to use such transferred materials and/or information solely for research and not for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic, or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. If the purchaser is unwilling to accept the limitations of this limited use statement, InvivoGen is willing to accept return of the product with a full refund. The product must be returned in resaleable condition. For information on purchasing a license to this product for purposes other than research, contact InvivoGen, 3950 Sorrento Valley Blvd. Suite 100, San Diego, CA Tel: Fax: TECHNICAL SUPPORT InvivoGen USA (Toll Free): InvivoGen USA (International): +1 (858) InvivoGen Europe: +33 (0) InvivoGen Hong Kong: info@invivogen.com

2 pdrive5lucia-hnphs1 A plasmid encoding the human nephrin (hnphs1) promoter Catalog # pdrive5lc-hnphs For research use only Version # 16E03-MM PRODUCT INFORMATION Contents: 20 µg of pdrive5lucia-hnphsi provided as lyophilized DNA 4 pouches of E. coli Fast-Media Zeo (2 TB and 2 Agar) Storage and Stability: Product is shipped at room temperature. Lyophilized DNA should be stored at -20 C. Resuspended DNA should be stored at -20 C and is stable for up to 1 year. Store E. coli Fast-Media at room temperature in a dry and cool place. Fast-Media pouches are stable for 2 years when stored properly. Quality Control: Plasmid construct has been confirmed by restriction analysis and full length ORF sequencing. Plasmid DNA was purified by ion exchange chromatography. GENERAL PRODUCT USE pdrive is an expression plasmid containing a native or composite promoter of interest. pdrive may be used to: - Subclone a promoter of interest into another vector. Unique restriction sites are present at each end of the promoter allowing convenient excision. The 5 sites are SdaI and SpeI. SdaI is compatible with NsiI and PstI. SpeI is compatible with AvrII, NheI and XbaI. The 3 restriction site is BspHI which includes the ATG start codon, and is compatible with NcoI and BspLU11I. - Compare the activity of different promoters in transient transfection experiments. Each pdrive5lucia promoter drives the expression of the Lucia luciferase reporter gene which allows for testing of the promoter s activity in transient transfection experiments. Furthermore, the Lucia luciferase gene is flanked by unique restriction sites (BspHI and NheI) for easy replacement with a different gene of interest. PROMOTER CHARACTERISTICS Human Nephrin (hnphs1) promoter Complete promoter size: 1233 bp Specificity: Podocytes Nephrin is a transmembrane protein that belongs to the immunoglobulin superfamily and is localized to the slit diaphragms that are a critical component of the glomerular filtration barrier in the kidney. A 1.25 kb fragment of the nephrin gene promoter, which includes the initiation codon and immediate 5 -flanking region, is capable of directing podocytespecific expression of a b-galactosidase transgene in vivo 1,2. This promoter lacks a TATA box but contains GATA binding sites and E-box consensus sequences. An enhancer was identified within the proximal promoter which is activated by the Wilms tumor suppressor protein (WT1) in vitro Wong MA. et al., Identification and characterization of a glomerularspecific promoter from the human nephrin gene. Am J Physiol Renal Physiol. 279(6):F Moeller MJ. et al., Two gene fragments that direct podocyte-specific expression in transgenic mice. J Am Soc Nephrol. 13(6): Guo G. et al., WT1 activates a glomerular-specific enhancer identified from the human nephrin gene. J Am Soc Nephrol. 15(11): TECHNICAL SUPPORT InvivoGen USA (Toll Free): InvivoGen USA (International): +1 (858) InvivoGen Europe: +33 (0) InvivoGen Hong Kong: info@invivogen.com PLASMID FEATURES Lucia luciferase is a synthetic CpG-free gene that codes for a secreted coelenterazine-utilizing luciferase. ORF size (from the ATG to the stop codon): 634 bp. Lucia luciferase activity can be evaluated using QUANTI-Luc, an assay reagent containing all the components required to quantitively measure the activity of Lucia luciferase and other coelenterazine-utilizing luciferases. SV40 pan: The Simian Virus 40 late polyadenylation signal enables efficient cleavage and polyadenylation reactions resulting in high levels of steady-state mrna. pmb1 Ori is a minimal E. coli origin of replication with the same activity as the longer Ori. EM2K is a bacterial promoter that enables the constitutive expression of the antibiotic resistance gene in E. coli. Zeo gene confers Zeocin resistance therefore allowing the selection of transformed E. coli carrying a pdrive plasmid. Note: Stable transfection of clones cannot be performed due to the absence of an eukaryotic promoter upstream of the Sh ble gene. METHODS Plasmid resuspension: Quickly spin the tube containing the lyophilized plasmid to pellet the DNA. To obtain a plasmid solution at 1 µg/µl, resuspend the DNA in 20 µl of sterile H 2 O. Store resuspended plasmid at -20 C. Plasmid amplification and cloning: Plasmid amplification and cloning can be preformed in E. coli GT116 other commonly used laboratory E. coli strains, such as DH5a. Selection of bacteria with E. coli Fast-Media Zeo: E. coli Fast-Media Zeo is a fast and convenient way to prepare liquid and solid media for bacterial culture by using only a microwave. E. coli Fast-Media Zeo is a TB (liquid) or LB (solid) based medium with zeocin, and contains stabilizers. E. coli Fast-Media Zeo can be ordered separately (catalog code fas-zn-l, fas-zn-s). Method: 1- Pour the contents of a pouch into a clean borosilicate glass bottle or flask. 2- Add 200 ml of distilled water to the flask 3- Heat in a microwave on MEDIUM power setting (about 400 Watts), until bubbles start appearing (approximately 3 minutes). Do not heat a closed container. Do not autoclave Fast-Media. 4- Swirl gently to mix the preparation. Be careful, the bottle and media are hot, use heatproof pads or gloves and care when handling. 5- Reheat the media for 30 seconds and gently swirl again. Repeat as necessary to completely dissolve the powder into solution. But be careful to avoid overboiling and volume loss. 6- Let agar medium cool to 45 C before pouring plates. Let liquid media cool to 37 C before seeding bacteria. Note: Do not reheat solidified Fast-Media as the antibiotic will be permanently destroyed by the procedure.

3 SdaI (29) EcoRI (19) XbaI (15) NotI (2) SpeI (36) NcoI (239) ori pmb1 SacI (620) hnephrin prom SgrAI (2815) EM2K Zeo pdrive5lucia-hnphs1 (3812 bp) Lucia BspHI (1271) SspI (2387) SV40 pan BglII (1389) NheI (1908) EcoRV (1686) 100

4 EcoRI (19) NotI (2) XbaI (15) SdaI (29) SpeI (36) GCGGCCGCTATGCATCTAGAATTCCTGCAGGGCCCACTAGTCTGTAATCCCAGCATTTTGGGAGGCTGAGGCAGATGGATCACCTGAGGTCAGGAGTTCG AGACCAGCCTGGCCAACATGATGAAACCCCGTCTCTAGTAAAAATACAAAAATTAGCCAGGCATGGTGCTATATACCTGTAGTACCAGCTACTTGGGAGA NcoI (239) CAGAGGTGGGAGAATTACTTGAACCTGGGAGGTTCAAGCCATGGGAGGTGGAAGTTGCAGTGAGCCGAGATGCCACTGCACTCCAGCCTGAGCAACAGAG CAAGACTATCTCAAGAAAAAAAAGAAAGAAAGAAAGGGACTTGCCAAGGTCATGTATCAGGGCAAGGAAGAGCTGGGGGCCCAGCTGGCTGCTCCCCTGC TGAGCTGGGAGACCACCTTGATCTGACTTCTCCCATCTTCCCAGCCTAAGCCAGGCCCTGGGGTCACGGAGGCTGGGGAGGCACCGAGGAACGCGCCTGG CATGTGCTGACAGGGAATTTTATGCTCCAGCTGGGCCAGCTGGGAGGAGCCTGCTGGGCAGAGGCCAGAGCTGGGGGCTCTGGAAGGTACCTGGGGGAGG SacI (620) TTGCACTGTGAGAATGAGCTCAAGCTGGGTCAGAGAGCAGGGCTGACTCTGCCAGTGCCTGCATCAGCCTCATCGCTCTCCTAGGCTCCTGGCCTGCTGG ACTCTGGGCTGCAGGTCCTTCTTGAAAGGCTGTGAGTAGTGAGACAAGGAGCAGGAGTGAGGGGTGGCAGGAGAGAAGATAGAGATTGAGAGAGAGAGAG AGAGAGACAGAGAGAGAGGAAGAGACAGAGACAAAAGGAGAGAGAACGGCTTAGACAAGGAGAGAAAGATGGAAAGATAAAGAGACTGGGCGCAGTGGCT CACGCCTGTAATCCCAACACTTGGGGAGGCCAAGGTGGGAGGATGGCTTGAAGGAAAGAGTCTGAGATCAACCTGGCCAACATAGTGAGACCCCGTCTCT AAAAAAAAAAAAGAAAAAAAAAAGAAAAAAGAAAAAAAAGTTTTTTTAAAGAGACAGAGAAAGAGACTCAGAGATTGAGACTGAGAGCAAGACAGAGAGA GACACTCACAGGGAAGAGGGGAAGAGGAAAACGAGAAAGGGAGGAGAGTAACGGAAAGAGATAAAAAAGAAAAGCAGGTGGCAGAGACACAGAGAGAGGG BspHI (1271) 1201 ACCCAGAGAAAGCCAGACAGACGCAGGTGGCTGGCAGCGGGCGCTGTGGGGGTCACAGTAGGGGGACCTGTcATGATGGAAATCAAGGTGCTGTTTGCCC 1 M M E I K V L F A BglII (1389) 1301 TCATCTGTATTGCTGTTGCTGAGGCAAAACCCACTGAAATCAATGAAGACCTCAATATAGCTGCTGTGGCCTCCAACTTTGCCACCACAGATCTTGAGAC 10 L I C I A V A E A K P T E I N E D L N I A A V A S N F A T T D L E T 1401 TGACCTGTTCACCAACTGGGAGACCATGAATGTGATTAGCACTGACACAGAGCAGGTGAACACAGATGCTGACAGGGGCAAGCTGCCTGGCAAAAAACTC 43 D L F T N W E T M N V I S T D T E Q V N T D A D R G K L P G K K L 1501 CCCCCAGATGTCCTGAGGGAGCTGGAGGCCAATGCCAGAAGGGCTGGTTGCACAAGAGGCTGCCTCATTTGCCTCTCCCACATTAAGTGCACCCCTAAGA 77 P P D V L R E L E A N A R R A G C T R G C L I C L S H I K C T P K EcoRV (1686) 1601 TGAAGAAATTTATCCCTGGCAGGTGCCACACTTATGAAGGTGAAAAGGAGTCTGCTCAGGGAGGGATTGGAGAGGCAATTGTTGATATCCCAGAGATTCC 110 M K K F I P G R C H T Y E G E K E S A Q G G I G E A I V D I P E I P 1701 TGGCTTCAAGGATAAGGAGCCACTGGACCAGTTTATTGCTCAAGTGGACCTCTGTGCTGATTGCACCACTGGCTGTCTGAAGGGCCTTGCCAATGTCCAG 143 G F K D K E P L D Q F I A Q V D L C A D C T T G C L K G L A N V Q 1801 TGCTCTGACCTCCTGAAGAAGTGGCTTCCCCAGAGGTGTACCACTTTTGCCAGCAAGATTCAGGGTAGGGTGGACAAAATCAAGGGTCTGGCTGGGGACA 177 C S D L L K K W L P Q R C T T F A S K I Q G R V D K I K G L A G D NheI (1908) 1901 GATGATAGCTAGCTGGCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGT 210 R 2001 GATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGG 2101 AGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGAATTAATTCTAAAATACAGCATAGCAAAACTTTAACCTCCAAATCAAGCCTCTACTTG AATCCTTTTCTGAGGGATGAATAAGGCATAGGCATCAGGGGCTGTTGCCAATGTGCATTAGCTGTTTGCAGCCTCACCTTCTTTCATGGAGTTTAAGATA SspI (2387) TAGTGTATTTTCCCAAGGTTTGAACTAGCTCTTCATTTCTTTATGTTTTAAATGCACTGACCTCCCACATTCCCTTTTTAGTAAAATATTCAGAAATAAT TTAAATACATCATTGCAATGAAAATAAATGTTTTTTATTAGGCAGAATCCAGATGCTCAAGGCCCTTCATAATATCCCCCAGTTTAGTAGTTGGACTTAG GGAACAAAGGAACCTTTAATAGAAATTGGACAGCAAGAAAGCGAGCTTCTAGCTTATCCTCAGTCCTGCTCCTCTGCCACAAAGTGCACGCAGTTGCCGG 125 D Q E E A V F H V C N G A 2601 CCGGGTCGCGCAGGGCGAACTCCCGCCCCCACGGCTGCTCGCCGATCTCGGTCATGGCCGGCCCGGAGGCGTCCCGGAAGTTCGTGGACACGACCTCCGA 111 P D R L A F E R G W P Q E G I E T M A P G S A D R F N T S V V E S 2701 CCACTCGGCGTACAGCTCGTCCAGGCCGCGCACCCACACCCAGGCCAGGGTGTTGTCCGGCACCACCTGGTCCTGGACCGCGCTGATGAACAGGGTCACG 78 W E A Y L E D L G R V W V W A L T N D P V V Q D Q V A S I F L T V SgrAI (2815) 2801 TCGTCCCGGACCACACCGGCGAAGTCGTCCTCCACGAAGTCCCGGGAGAACCCGAGCCGGTCGGTCCAGAACTCGACCGCTCCGGCGACGTCGCGCGCGG 44 D D R V V G A F D D E V F D R S F G L R D T W F E V A G A V D R A T 2901 TGAGCACCGGAACGGCACTGGTCAACTTGGCCATGATGGCTCCTCCTGTCAGGAGAGGAAAGAGAAGAAGGTTAGTACAATTGCTATAGTGAGTTGTATT 11 L V P V A S T L K A M 3001 ATACTATGCAGATATACTATGCCAATGATTAATTGTCAAACTAGGGCTGCAGGTTAATTAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCG TAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGA CTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGG GAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCC CGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGA

5 GCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAG TTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAA AAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGGCTAGTTAATTA ACATTTAAATCA