SAN TA C RUZ BI OTEC HNOL OG Y, INC . PTRF shRNA (m) Lentiviral Particles: sc-76294-V BACKGROUND APPLICATIONS The termination of transcription by RNA polymerase I (Pol I) requires the PTRF shRNA (m) Lentiviral Particles is recommended for the inhibition of involvement of several proteins, including TTF-1 (thyroid transcription factor-1) PTRF expression in mouse cells. which pauses transcription, thus allowing the Pol I complex to dissociate and release the subsequent pre-rRNA. PTRF (polymerase I and transcript release SUPPORT REAGENTS factor), also known as FKSG13, is a 390 amino acid protein that is required Control shRNA Lentiviral Particles: sc-108080. Available as 200 µl frozen for the dissociation of the transcription complex. Localized to various places viral stock containing 1.0 x 10 6 infectious units of virus (IFU); contains an within the cell, including the cell membrane, microsome, nucleus and cyto - shRNA construct encoding a scrambled sequence that will not lead to the plasm, PTRF binds the 3' end of pre-rRNA while simultaneously interacting specific degradation of any known cellular mRNA. with Pol I and TFF-1, thus allowing the Pol I complex to release from the template. Three isoforms of PTRF are expressed due to alternative splicing GENE EXPRESSION MONITORING events. PTRF (4a): sc-517589 is recommended as a control antibody for monitoring REFERENCES of PTRF gene expression knockdown by Western Blotting (starting dilution 1:200, dilution range 1:100-1:1000) or immunofluorescence (starting dilution 1. Mason, S.W., et al. 1997. Identification of a tran script release activity 1:50, dilution range 1:50-1:500). acting on ternary transcription complexes containing murine RNA poly - merase I. EMBO J. 16: 163-172. To ensure optimal results, the following support reagents are recommended: 1) Western Blotting: use m-IgG κ BP-HRP: sc-516102 or m-IgG κ BP-HRP (Cruz 2. Jansa, P., et al. 1998. Cloning and functional characterization of PTRF, a Marker): sc-516102-CM (dilution range: 1:1000-1:10000), Cruz Marker™ novel protein which induces dissociation of paused ternary transcription Molecular Weight Standards: sc-2035, UltraCruz ® Blocking Reagent: complexes. EMBO J. 17: 2855-2864. sc-516214 and Western Blotting Luminol Reagent: sc-2048. 2) Immunofluo- 3. Hasegawa, T., et al. 2000. PTRF (polymerase I and transcript-release rescence: use m-IgG κ BP-FITC: sc-516140 or m-IgG κ BP-PE: sc-516141 factor) is tissue-specific and interacts with the BFCOL1 (binding factor (dilution range: 1:50-1:200) with UltraCruz ® Mounting Medium: sc-24941 of a type-I collagen promoter) zinc-finger transcription factor which or UltraCruz ® Hard-set Mounting Medium: sc-359850. binds to the two mouse type-I collagen gene promoters. Biochem. J. 347: 55-59. RT-PCR REAGENTS 4. Jansa, P., et al. 2001. The transcript release factor PTRF augments riboso - Semi-quantitative RT-PCR may be performed to monitor PTRF gene expression mal gene transcription by facilitating reinitiation of RNA polymerase I. knockdown using RT-PCR Primer: PTRF (m)-PR: sc-76294-PR (20 µl). Annealing Nucleic Acids Res. 29: 423-429. temperature for the primers should be 55-60° C and the extension temperature should be 68-72° C. 5. Aboulaich, N., et al. 2004. Vectorial proteomics reveal targeting, phos - phorylation and specific fragmentation of polymerase I and transcript BIOSAFETY release factor (PTRF) at the surface of caveolae in human adipocytes. Biochem. J. 383: 237-248. Lentiviral particles can be employed in standard Biosafety Level 2 tissue culture facilities (and should be treated with the same level of caution as CHROMOSOMAL LOCATION with any other potentially infectious reagent). Lentiviral particles are repli- cation-incompetent and are designed to self-inactivate after transduction Genetic locus: Ptrf (mouse) mapping to 11 D. and integration of shRNA constructs into genomic DNA of target cells. PRODUCT RESEARCH USE PTRF shRNA (m) Lentiviral Particles is a pool of concentrated, transduction- The purchase of this product conveys to the buyer the nontransferable right ready viral particles containing 3 target-specific constructs that encode to use the purchased amount of the product and all replicates and derivatives 19- 25 nt (plus hairpin) shRNA designed to knock down gene expression. Each for research purposes conducted by the buyer in his laboratory only (whether 6 vial contains 200 µl frozen stock containing 1.0 x 10 infectious units of virus the buyer is an academic or for-profit entity). The buyer cannot sell or other - (IFU) in Dulbecco’s Modified Eagle’s Medium with 25 mM HEPES pH 7.3. wise transfer (a) this product (b) its components or (c) materials made using Suitable for 10-20 transductions. Also see PTRF siRNA (m): sc-76294 and this product or its components to a third party, or otherwise use this product PTRF shRNA Plasmid (m): sc-76294-SH as alternate gene silencing products. or its components or materials made using this product or its components for Commercial Purposes. STORAGE Store lentiviral particles at -80º C. Stable for at least one year from the PROTOCOLS date of shipment. Once thawed, particles can be stored at 4º C for up to See our web site at www.scbt.com for detailed protocols and support one week. Avoid repeated freeze thaw cycles. products. Santa Cruz Biotechnology, Inc. 1.800.457.3801 831.457.3800 fax 831.457.3801 Europe +00800 4573 8000 49 6221 4503 0 www.scbt.com.