Sirna cassette protocol

GenScript Adenoviral Vector-based siRNA
Protocol

Technical Manual No. 0165 Version 04032007

Introduction ………………………………………………………………………………. Vector-based siRNA …………………………………………………………………… Adenoviral siRNA Vector ………………………………………………………………. Product description ….…………………………………………………………………… Cloning siRNA insert into GenScript pRNA vectors ………………………………… Producing adenoviral particles and infecting mammalian cells ……………………. VII References .…………………………………………………………………………….
I. Introduction:
RNAi (RNA interference) is a phenomenon that small double-stranded RNA (referred as small interfering RNA
or siRNA) can knock down the expression of its corresponding gene. RNAi has been observed in plants,
C.elegans and Drosophila long time ago. It was until recently that RNAi was discovered to work in mammalian
system
Small interfering RNA (siRNA) is 19-22 nt double-stranded RNA. It works by cleaving and destroying its cognate
RNA. siRNA first assembles into RNA-induced silencing complexes (RISCs), where it then activates the
complex by unwinding its RNA strands. The unwound RNA strands subsequently guide the complex to the
complementary RNA molecules, where the complex cleaves and destroys the cognate RNA, which results in
RNAi phenomenon. RNAi has evolved into a powerful tool to study gene functions.
II. Vector-based siRNA
Using DNA vector-based siRNA technology, a small DNA insert (about 70 bp) encoding a short hairpin RNA targeting the gene of interest is cloned into a commercially available vector. The insert-containing vector can be transfected into the cell, and it expresses the short hairpin RNA. The hairpin RNA is rapidly processed by the cellular machinery into 19-22 nt double stranded RNA (siRNA). Key features of vector-based siRNA: 1. More stable and easier to handle: Vector-based siRNA is delivered as plasmids, which is more stable
and easier to handle than synthetic siRNA. 2. Stable cell line can be established: Vector based siRNA allows you to obtain a stable cell line, and
observe long-term effects of RNAi [2-5]. 3. Viral siRNA can be preparedViral siRNA can be used to infect primary cell lines and used for
4. Inducible system can be established: Vector based siRNA allows you to establish an inducible
system by using a vector with an inducible promoter. 5. A knock-out mouse line can be established using transgenic siRNA method
6. Unlimited supply: once a DNA construct is made, you will have unlimited supply of siRNA.
GenScript Corporation Tel: 732-885-9188 Fax: 732-210-0262 www.genscript.com email: info@genscript.com GenScript Adenoviral Vector-based siRNA Protocol It is recommended that at least 3 vector-based siRNA should be prepared for each gene to find the most potent and most specific siRNA. Here are the reasons: 1. Not all siRNA target sequences are equally potent: Because of secondary structure and other
factors, some target sequences are more potent than others. It is better to test at least three vector-siRNA constructs to find the most potent one. 2. Not all siRNA silencing effects are gene-specific: It has been reported that some siRNA silencing
effects are not gene-specific because of various reasons. It is better to validate your experiments results using three vector-based siRNA constructs. 3. Results from synthetic siRNA or siRNA cassette cannot be completely transferred to vector-
based siRNA construct: Vector-based siRNA is different from synthetic siRNA oligos or siRNA
cassette. Although the results from synthetic siRNA oligos or siRNA cassette can suggest the most
potent siRNA targets, the results cannot be completely duplicated in vector-based siRNA for unknown
reasons.
4. The experiment is still the gold test stone: Although we are proud of our vector-based siRNA design
program, the best design is still not as good as what the experiments can tell you. III. Adenoviral siRNA Vector

Adenoviruses are linear, double stranded DNA viruses capable of infecting a majority of human and many
nonhuman cell types including mouse, rat, dog, chicken, rabbit, sheep, pig, and nonhuman primates, and
infection is not dependent on active host cell division. Adenoviral vectors have two outstanding features: high
efficiency gene delivery and high-level gene expression. Due to these attributes, adenoviral vectors can be
used for overexpressing siRNAs in a broad range of mammalian cells. The adenoviral vectors developed at
GenScript have proved to be high efficient for siRNA expression in mammalian cells [9].
GenScript has developed two kinds (named differently) of adenoviral shuttle siRNA vectors:
Shuttle vectors. These adenoviral shuttle vectors are compatible with Clontech Adeno-X™
Expression System 1 (Cat. No. 631513)
(http://orders.clontech.com/AIT/Ecommerce/Clontech/ProductCatalog.aspx?item=492574). These
vectors are: pRNA-U6.1/Shuttle (SD1205), pRNA-H1.1/Shuttle (SD1206), pRNAT-H1.1/Shuttle
(SD1216), and pRNAin-H1.2/Shuttle (SD1234), they use U6 or H1 or inducible H1 promoter to drive
the siRNA expression.
Adeno vectors. GenScript adenoviral shuttle vectors are compatible with Stratagene AdEasy
Adenoviral (http://www.stratagene.com/products/displayProduct.aspx?pid=86) Vector System.
These vectors are: pRNA-H1.1/Adeno (SD1209), pRNAT-H1.1/Adeno (SD1219) and pRNATin-
H1.2/Adeno (SD1229), they use H1 or inducible H1 promoter to drive the siRNA expression.
These vectors contain elements that allow recombination of the siRNA construct into adenoviral genome. The adenoviral particles are then used to infect mammalian cells to express siRNA in the host cells. U6, H1 promoter (H1.1) and inducible H1 promoter (H1.2) are used to drive the siRNA expression. The H1.2 promoter is an engineered inducible H1 promoter containing a tetracycline operator (TetO1). The tetracycline operator itself has no effect on expression, in the absence of the tetracycline repressor (TetR), H1.2 just behaves like H1. When the tetracycline repressor (TetR) is present, it effectively binds the TetO1 and blocks transcription. In the presence of tetracycline or doxycycline, the inducer binds TetR and causes the TetR protein to release the TetO1 site, and derepresses the transcription from H1 promoter. These vectors are designed for mammalian transfection. These vectors also have the following features: Three of the vectors (SD1216, SD1219 and SD1229) carry a GFP (coral GFP) marker under CMV promoter* control for tracking the transfection efficiency and viral infection. siRNA effect can be tested first for siRNA optimization before you produce adenoviral particles and infect target cells. After cloning the siRNA construct into the vectors, you can test the siRNA effect by transfection of the siRNA-containing vectors into the mammalian cells. H1 will drive the siRNA expression. GenScript Corporation Tel: 732-357-9188 Fax: 732-210-0262 www.genscript.com email: info@genscript.com GenScript Adenoviral Vector-based siRNA Protocol
The circle maps of all these vectors:
SD1205: pRNA-U6.1/Shuttle SD1206: pRNA-H1.1/Shuttle

SD1216: pRNAT-H1.1/Shuttle
SD1234: pRNAin-H1.2/Shuttle
GenScript Corporation Tel: 732-357-9188 Fax: 732-210-0262 www.genscript.com email: info@genscript.com GenScript Adenoviral Vector-based siRNA Protocol
SD1209: pRNA-H1.1/Adeno
SD1219: pRNAT-H1.1/Adeno

SD1229: pRNATin-H1.2/Adeno

Important points for consideration before using Inducible vector:

1. H1.2 promoter only works as an inducible promoter in the cells containing tetracycline repressor (TetR). In the cells without TetR, H1.2 behaves as a regular H1 promoter. 2. For inducible experiments, the serum is very critical. As FBS from most vendors contains Tetracycline itself, it is very critical to use Tetracycline free FBS. We recommend to use Clontech Tet approved FBS (Clontech, Cat#631101). 3. Either tetracycline or doxcycline can be used as an inducer reagent. The concentration for tetracycline recommended is 1 g/ml to 5 g/ml, and the concentration for doxcycline recommended is 1 g/ml. 4. Tetracycline or doxycycline should be added to the medium right after transfection. The induction time GenScript Corporation Tel: 732-357-9188 Fax: 732-210-0262 www.genscript.com email: info@genscript.com GenScript Adenoviral Vector-based siRNA Protocol
IV. Product Description

Cat. No.: SD1205, SD1206, SD1216, SD1234, SD1209, SD1219 and SD1229.
Description: A small DNA insert (about 70-80 bp) encoding a short hairpin RNA targeting the gene of interest is
cloned into a shuttle siRNA expression vector at the multiple cloning site (MCS). The insert-containing vector
can be transfected into the cell, and Promoter: Options include human U6 or H1 promoter.
 Term: Poly(T) termination signal.  Coral GFP (cGFP) marker to track the transfection efficiency and viral infection.  MCS is used to clone the DNA insert into GenScript pRNA vectors. Quantity: 10 µg (MiniPrep) or 100 µg (MaxiPrep).
Quality Control: The insert is sequencing verified, and trace data is provided together with the plasmid.
Storage: -20 °C after receiving.
GenScript Adenoviral siRNA Expression Vectors:

Promoter Human U6 Human H1 Human H1 Human inH1 Human H1 Human H1 Human inH1

V. Cloning siRNA insert into GenScript pRNA vectors.

1. siRNA Design: GenScript has developed a proprietary algorithm for designing a siRNA target and building the insert. To find the target sequence and build siRNA insert, please use GenScript’s siRNA design center siRNA Target Finder and siRNA Construct Builder (http://www.genscript.com/rnai.html). 2. Custom vector-based siRNA constructs: GenScript provides custom vector-based siRNA at a very competitive price. Alternatively, you can use the following protocol to make the vector-based siRNA constructs yourself. 3. Order two oligos with appropriate restriction sites. The oligos must be PAGE purified oligos. Dissolve the oligos in water to 1 μg/μl concentration. 4. Anneal the two oligos. Prepare a 20 μl annealing reaction in the following way: 1 μl top-strand oligo 1 μl bottom-strand oligo 1 μl 20 x SSC (Sigma, Cat. S6639) 17 μl water 5. Heat the mixture to 95 oC for 10 min. Take it out and put it at room temperature for one hour. Dilute the mixture to a final concentration of 40 ng/μl. 6. Cut the vector with appropriate restriction enzymes. Run 1% agarose gel and purify the vector. 7. Ligate the vector with the insert using T4 ligase (the molar ratio of insert to vector is 3:1). 8. Transform the ligation mixture into competent DH5a cells (Invitrogen, Cat. 18258-012). Plate it on LB- amp plates. For SD1209, SD1219 and Sd1229, transform the ligation mixture into One Shot Stbl3 competent cells (Invitrogen, Cat. C7373-03) and plate it on LB-kan plates (Kanamycin at 25 μg/ml). 9. Choose at least 15 clones and grow them. Prepare MiniPreps from culture using GenScript MiniPrep kit. 10. Cut the plasmids with appropriate restriction enzymes. Run 3% gel to check whether the plasmids have 11. Sequence the positive clones to verify the sequence of the insert. GenScript Corporation Tel: 732-357-9188 Fax: 732-210-0262 www.genscript.com email: info@genscript.com GenScript Adenoviral Vector-based siRNA Protocol
VI. Producing Adenoviral Particles and Infecting Mammalian Cells.

A. Using shuttle vectors that are compatible with Clontech Adeno-X expression system:
To deliver the siRNA expression system into mammalian cells using GenScript shuttle vectors, you also need to
order BD Adeno-X Expression System 1 (Cat. No. 631513) from BD Biosciences Clontech.
A brief procedure to express your siRNA in the host cell line. For more details, please follow Clontech
Retro-X™ System Manual to produce adenoviral particles and infect host cells.
1. Clone your siRNA construct into GenScript shuttle vectors using appropriate restriction enzymes.
You can use U6 promoter, H1 promoter or inducible H1 promoter for your siRNA expression. 2. Digest shuttle vectors with PI-Sce I and I-Ceu I, ligate the fragment containing the siRNA to the 3. Transform E. coli strain DH5α to select for and amplify recombinant clones. 4. Transfect a low-passage HEK 293 cell line with the recombinant adenoviral DNA and harvest 5. Infect your host cells by adding the viral supernatant. 6. Use appropriate methods to analyze siRNA silencing effect.
B. Using adeno vectors that are compatible with Stratagene AdEasy Adenoviral Vector System:
To deliver the siRNA expression system into mammalian cells using GenScript adeno vectors, you also need to
order Stratagene pAdEasy-1 vector (Catalog#240005), BJ5183 competent cells (Catalog#200154), AD-293 cells
(Catalog#240085) and MBS Mammalian Transfection Kit (Catalog#200388).
A brief procedure to express your siRNA in the host cell line. For more details, please follow Stratagene
AdEasy Adenoviral Vector System Manual to produce adenoviral particles and infect host cells.
1. Clone your siRNA construct into GenScript adeno vectors using appropriate restriction enzymes. You
can use either H1 promoter or inducible H1 promoter for your siRNA expression. 2. Contransform the resultant plasmid into E. coli strain BJ5183 with the adenoviral backbone plasmid pAdEasy-1. Recombinant adenoviral plasmids are selected on kanamycin and confirmed by restriction digest. 3. Transfect AD-293 cells with the recombinant adenoviral plasmid and harvest recombinant adenovirus a 4. Infect your host cells by adding the viral supernatant. 5. Use appropriate methods to analyze siRNA silencing effect.
VII. References
1. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494-498. 2. Yu JY, DeRuiter SL, Turner DL. (2002) RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci U S A. 99(9):6047-6052. 3. Brummelkamp, T.R., Bernards, R., and Agami, R. (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296: 550-553. 4. Jacque, J.-M., Triques, K., and Stevenson, M. (2002) Modulation of HIV-1 replication by RNA 5. Sui, G., Soohoo, C., Affar, E.B., Gay, F., Shi, Y., Forrester, W.C., and Shi, Y. (2002) A DNA vector- based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl. Acad. Sci. USA 99(8): 5515-5520. 6. Shen C, Buck AK, Liu X, Winkler M, and Reske SN. (2003) Gene silencing by adenovirus-delivered 7. Barton GM, and Medzhitov R. (2002) Retroviral delivery of small interfering RNA into primary cells. Proc GenScript Corporation Tel: 732-357-9188 Fax: 732-210-0262 www.genscript.com email: info@genscript.com GenScript Adenoviral Vector-based siRNA Protocol 8. Kunach T, Gish G, Lickert H, Jones N, Pawson T, and Rossant J. (2003) Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype. Nature Biotechnology 21:559-561. 9. Jiliang Zhou and B. Paul Herring. Mechanisms responsible for the promoter specific effects of myocardin. J. Biol. Chem. Jan 2005; 10.1074/jbc.M411586200

Limited Use Label License

This product is the subject of the following patents: U.S. Patent No. 6,573,099 and foreign counterparts co-
owned by Benitec Australia Ltd. Not-for-profit and academic entities who purchase this product receive the
non-transferable right to use the product and its components in non-Commercial Purpose internal research.
All commercial and for-profit purchasers using this product for a Commercial Purpose are required to execute a
commercial research license with Benitec by contacting Benitec at licensing@benitec.com . Entities wishing to
use ddRNAi as a therapeutic agent or as a method to treat/prevent human disease also should contact Benitec
at licensing@benitec.com .
Commercial Purpose means any activity by a party for tangible consideration and may include, but is not limited to, (i) use of the product in manufacturing; (ii) use of the product to provide services, information or data for a compensatory fee; (iii) use of the product for therapeutic, diagnostic or prophylactic purposes; (iv) use of the product in drug discovery or target validation and/or (v) resale of the product or its components whether or not the product is resold for use in research. If purchaser is not willing to accept the limitations of this label license, seller is willing to accept the return of the product with a full refund. For terms and additional information relating to a commercial research license, or for licensing for any human therapeutic purpose, contact Benitec by email a. * Limited Use Label License: The use of CMV promoter is covered under U. S. Patent No. 5,168,062 and 5,385,839 owned and licensed by the University of Iowa Research Foundation and is sold for research use only. Commercial users must obtain a license to these patents directly from the University of Iowa Research Foundation (UIRF), 214 Technology Innovation Center, Iowa City, Iowa 52242. For further information, please contact the Associate Director of UIRF, at 319-335-4546.
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