VEGF siRNA Delivery System Using Arginine-Grafted
Bioreducible Poly(disul?de amine)
Sun Hwa Kim,?Ji Hoon Jeong,?Tae-il Kim,?Sung Wan Kim,?and
David A.Bull*,§
Center for Controlled Chemical Deli V ery,Department of Pharmaceutics and Pharmaceutical Chemistry,Uni V ersity of Utah,Salt Lake City,Utah84112,College of
Pharmacy,Sungkyunkwan Uni V ersity,Suwon440-746,South Korea,and Di V ision of
Cardiothoracic Surgery,School of Medicine,Uni V ersity of Utah Health Sciences Center,
Salt Lake City,Utah84132
Received September10,2008;Revised Manuscript Received November17,2008;Accepted
November18,2008
Abstract:Small interfering RNAs(siRNAs)are able to silence their target genes when they are successfully delivered intact into the cytoplasm.Delivery systems that enhance siRNA localization to the cytoplasm can facilitate gene silencing by siRNA therapeutics.We describe an arginine-conjugated poly(cystaminebisacrylamide-diaminohexane)(poly(CBA-DAH-R)),a bioreducible cationic polymer,as an siRNA carrier for therapeutic gene silencing for cancer. After intracellular uptake of the siRNA/poly(CBA-DAH-R)polyplexes,the reductive environment of the cytoplasm cleaves the disul?de linkages in the polymeric backbone,resulting in decomplexing of the siRNA/poly(CBA-DAH-R)polyplexes and release of siRNA molecules throughout the cytoplasm.The siRNA/poly(CBA-DAH-R)polyplexes,which demonstrate increased membrane permeability with arginine modi?cation,have a similar level of cellular uptake as siRNA/bPEI polyplexes.The VEGF siRNA/poly(CBA-DAH-R)polyplexes,however,inhibit VEGF expression to a greater degree than VEGF siRNA/bPEI in various human cancer cell lines.The improved RNAi activity demonstrated by the VEGF siRNA/poly(CBA-DAH-R) polyplexes is due to enhanced intracellular delivery and effective localization to the cytoplasm of the VEGF siRNAs.These results demonstrate that the VEGF siRNA/poly(CBA-DAH-R) polyplex delivery system may useful for siRNA-based approaches for cancer therapy. Keywords:Cytoplasmic localization;siRNA;bioreducible cationic polymer;gene silencing; vascular endothelial growth factor;cancer therapy
Introduction
Synthetic siRNAs,21-23nucleotides in length,mediate target gene silencing by promoting mRNA degradation in the cytoplasm.1Since many diseases are caused by the inappropriate expression of disease-related genes,siRNA technology may provide new therapies for the treatment of diverse human diseases including cancer,obesity,heart disease,and diabetes.2,3siRNA therapeutics offer advantages over conventional pharmaceutics because of their powerful and speci?c gene silencing ability.The practical applications
*Corresponding author.Mailing address:Division of Cardiotho-racic Surgery,School of Medicine,University of Utah Health Sciences Center,Salt Lake City,UT84132.Phone:801-581-5311.Fax:801-585-3936.E-mail:david.bull@https://www.doczj.com/doc/b113130527.html,.?Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah.
?Sungkyunkwan University.
§University of Utah Health Sciences Center.(1)Hammond,S.M.;Caudy,A.A.;Hannon,G.J.Post-transcriptional
gene silencing by double-stranded RNA.Nat.Re V.Genet.2001, 2,110–119.
(2)McManus,M.T.;Haines,B.B.;Dillon,C.P.;Whitehurst,C.E.;
Van Parijs,L.;Chen,J.;Sharp,P.A.Gene silencing in mammals by small interfering RNAs.Nat.Re V.Genet.2002,3,737–747.
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nomics and potential as therapeutics.Nat.Re V.Drug Disco V ery 2004,3,318–329.
articles
718MOLECULAR PHARMACEUTICS VOL.6,NO.3,718–72610.1021/mp800161e CCC:$40.75 2009American Chemical Society
Published on Web12/04/2008
of siRNAs,however,have been limited by their instability and poor delivery to the necessary site of action.4The development of safe and effective delivery vehicles for siRNAs would greatly enhance the clinical utility of gene silencing siRNAs as a therapeutic modality for the treatment of cancer.
Various polycations have been developed as nonviral siRNA carriers to enhance the gene silencing activity of siRNA therapeutics.5,6Polycations can form highly stable polyelectrolyte complexes with siRNA molecules via elec-trostatic interactions between the positively charged poly-cations and the negatively charged phosphates of the nucleic acids.These polyelectrolyte formulations have been shown to improve the protection of siRNA against enzymatic degradation and increase the cellular uptake ef?ciency of siRNA.7,8The latter is important as suf?cient intact,func-tional siRNA must be localized in the cytoplasm to achieve an effective intracellular concentration of siRNA to silence the target mRNA.9
Bioreducible cationic polymers containing disul?de link-ages arranged regularly on the polymer backbone structure have been reported to be ef?cient gene delivery vehicles with minimal cellular toxicity.10,11The stable polyelectrolyte complexes composed of bioreducible cationic polymers and nucleic acids are readily degraded upon exposure to the reductive environment of the cytoplasm.12,13This unique characteristic of bioreducible cationic polymers leads to the release within the cytoplasm of nucleic acids(pDNA and siRNA).Unlike DNA transfection,delivery to the nucleus is not required for RNAi induction,due to the activity of siRNA in the cytoplasm.Therefore,polycations which are cleaved upon exposure to a reductive intracellular state offer the potential for controlled degradation in the cytoplasm, resulting in safe,high-ef?ciency siRNA delivery.
We have recently described several branched/linear biore-ducible cationic polymers,composed of low molecular weight cationic monomers and polymerized with disul?de linkages,as promising gene delivery vehicles.14-16The bioreducible cationic polymers form stable,nanosized poly-plexes with pDNA and siRNA and achieve higher transfec-tion ef?ciencies with signi?cantly less cytotoxicity compared to the conventional cationic polymer:branched polyethyl-enimine(bPEI).
To enhance the ef?cacy of therapeutic gene silencing,we synthesized an arginine-modi?ed polydisul?de poly(CBA-DAH-R)as an siRNA carrier for cancer gene therapy. Arginine residues are reported to enhance the cellular membrane permeability of many biologically active mol-ecules,including drugs,peptides,and plasmids.17-19The reductive degradation of the siRNA/poly(CBA-DAH-R) polyplexes was estimated from physicochemical measure-ments via dynamic light scattering and gel retardation analysis.The triggered release characteristics of the siRNA/ poly(CBA-DAH-R)polyplexes in correlation with cytoplas-mic siRNA localization and gene knockdown ef?ciency were investigated in human prostate carcinoma(PC-3cells).To verify that poly(CBA-DAH-R)could be a potential siRNA
(4)Oishi,M.;Nagasaki,Y.;Itaka,K.;Nishiyama,N.;Kataoka,K.
Lactosylated poly(ethylene glycol)-siRNA conjugate through acid-labile beta-thiopropionate linkage to construct pH-sensitive polyion complex micelles achieving enhanced gene silencing in hepatoma
cells.J.Am.Chem.Soc.2005,127,1624–1625.
(5)Li,S.D.;Huang,L.Targeted delivery of antisense oligodeoxy-
nucleotide and small interference RNA into lung cancer cells.Mol.
Pharmaceutics2006,3,579–588.
(6)Lee,S.H.;Choi,S.H.;Kim,S.H.;Park,T.G.Thermally sensitive
cationic polymer nanocapsules for speci?c cytosolic delivery and ef?cient gene silencing of siRNA:swelling induced physical disruption of endosome by cold shock.J.Controlled Release2008, 125,25–32.
(7)Zhang,S.;Zhao,B.;Jiang,H.;Wang,B.;Ma,B.Cationic lipids
and polymers mediated vectors for delivery of siRNA.J.
Controlled Release2007,123,1–10.
(8)Kim,S.H.;Jeong,J.H.;Lee,S.H.;Kim,S.W.;Park,T.G.
PEG conjugated VEGF siRNA for anti-angiogenic gene therapy.
J.Controlled Release2006,116,123–129.
(9)Lavigne,C.;Thierry,A.R.Speci?c subcellular localization of
siRNAs delivered by lipoplex in MCR-7breast cancer cells.
Biochimie2007,89,1245–1251.
(10)Gosselin,M.A.;Guo,W.;Lee,R.J.Ef?cient gene transfer using
reversibly cross-linked low molecular weight polyethylenimine.
Bioconjugate Chem.2001,12,989–994.
(11)Yockman,J.W.;Kastenmeier,A.;Erickson,H.M.;Brumbach,
J.G.;Whitten,M.G.;Albanil,A.;Li,D.Y.;Kim,S.W.;Bull,
D.A.Novel polymer carriers and gene constructs for treatment
of myocardial ischemia and infarction.J.Controlled Release(in press,available online6July2008).
(12)Jeong,J.H.;Christensen,L.V.;Yockman,J.W.;Zhong,Z.;
Engbersen,J.F.;Kim,W.J.;Feijen,J.;Kim,S.W.Reducible poly(amido ethylenimine)directed to enhance RNA interference.
Biomaterials2007,28,1912–1917.(13)Kim,S.H.;Jeong,J.H.;Ou,M.;Yockman,J.W.;Kim,S.W.;
Bull,D.A.Cardiomyocyte-targeted siRNA delivery by prostag-landin E2-Fas siRNA polyplexes formulated with reducible poly (amido amine)for preventing cardiomyocyte apoptosis.Bioma-terials2008,29,4439–4446.
(14)Christensen,L.V.;Chang,C.W.;Kim,W.J.;Kim,S.W.;Zhong,
Z.;Lin,C.;Engberson,J.F.;Feijen,J.Reducible poly(amido ethylenimine)s designed for triggered intracellular gene delivery.
Bioconjugate Chem.2006,17,1233–1240.
(15)Christensen,L.V.;Chang,C.W.;Yockman,J.W.;Conners,R.;
Jackson,H.;Zhong,Z.;Feijen,J.;Bull,D.A.;Kim,S.W.
Reducible poly(amido ethylenediamine)for hypoxia-inducible VEGF delivery.J.Controlled Release2007,118,254–261. (16)Ou,M.;Wang,X.L.;Xu,R.;Chang,C.W.;Bull,D.A.;Kim,
S.W.Novel biodegradable poly(disul?de amine)s for gene delivery with high ef?ciency and low cytotoxicity.Bioconjugate Chem.2008,19,626–633.
(17)Futaki,S.;Goto,S.;Sugiura,Y.Membrane permeability com-
monly shared among arginine-rich peptides.J.Mol.Recognit.
2003,16,260–264.
(18)Choi,J.S.;Nam,K.;Park,J.Y.;Kim,J.B.;Lee,J.K.;Park,
J.S.Enhanced transfection ef?ciency of PAMAM dendrimer by surface modi?cation with L-arginine.J.Controlled Release2004, 99,445–456.
(19)Kim,W.J.;Christensen,L.V.;Jo,S.;Yockman,J.W.;Jeong,
J.H.;Kim,Y.H.;Kim,S.W.Cholesteryl oligoarginine delivering vascular endothelial growth factor siRNA effectively inhibits tumor growth in colon adenocarcinoma.Mol.Ther.2006,14,343–350.
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delivery vehicle for target gene silencing in cancer cells, VEGF gene silencing by the poly(CBA-DAH-R)polyplexes with VEGF-targeted siRNAs was evaluated in four different human cancer cell lines(KB,HeLa,A2780,and A549). Experimental Section
Materials.Branched polyethylenimine(bPEI,M w25,000), dithiothreitol(DTT),3-(4,5-dimethyl-2-thiazolyl)-2,5-diphen-yl-2H-tetrazolium bromide(MTT),and DL-buthionine-sul-foxamine(BSO)were purchased from Sigma-Aldrich(St. Louis,MO).Poly(CBA-DAH-R)was prepared as shown in the supplemental?gure in the Supporting Information. Human VEGF siRNA(sense,5′-GGAGUACCCUGAUGA-GAUCdTdT-3′;antisense,5′-GAUCUCAUCAGGGUACUC-CdTdT-3′)and green?uorescent protein(GFP)siRNA (sense,5′-GCACGACUUCUUCAAGUCCdTdT-3′;anti-sense,5′-GGACUUGAAGAAGUCGUGCdTdT-3′)were synthesized,modi?ed and puri?ed by Dharmacon Co. (Lafayette,CO).For?ow cytometry and confocal micros-copy,siRNA was labeled with Cy3dye at the3′-terminal end of the sense strand.All cell culture products including fetal bovine serum(FBS),Roswell Park Memorial Institute 1640medium(RPMI1640),and Dulbecco′s phosphate buffered saline(PBS)were obtained from Invitrogen(Gibco BRL,Carlsbad,CA).
Dynamic Light Scattering(DLS)Assay.The siRNA polyplexes with poly(CBA-DAH-R)and bPEI were formed at various weight ratios(polymer/siRNA)ranging from10:1 to60:1and from0.1:1to60:1,respectively.A?xed amount of siRNA(3μg)was complexed with different amounts of polymers(poly(CBA-DAH-R)or bPEI)in0.4mL HEPES buffer(HEPES20mM,5%glucose,pH7.4).After30min incubation at room temperature,the polyplex solution was 6-fold diluted with deionized water.To determine the degradation ability of the siRNA/poly(CBA-DAH-R)poly-plexes,the siRNA polyplexes,which were formed with poly(CBA-DAH-R)and bPEI at a weight ratio of40:1and 1:1respectively,were further incubated with2.5mM DTT for2h at37°C.Particle sizes and surface charges were measured using a Zetasizer3000HS(Malvern Instrument, Inc.,Worcestershire,U.K.)at a wavelength of677nm with a constant angle of90°at25°C.
Electrophoretic Mobility Shift Assay.The siRNA/ poly(CBA-DAH-R)polyplexes were prepared at different weight ratios ranging from0:1to40:1.siRNA(0.3μg)was condensed with varying amounts of poly(CBA-DAH-R)in an aqueous phase(PBS30μL,pH7.4)and incubated at room temperature for30min.The siRNA/bPEI polyplexes were formed at a weight ratio of1:1.Each sample solution was loaded onto a2.0%agarose gel.Electrophoresis was carried out with a current of120V for15min in1×TAE buffer solution(10mM Tris/HCl,1%(v/v)acetic acid,1 mM EDTA).The retardation of the polyplexes was visualized with an image analyzer equipped with a UV transilluminator (GelDoc,BioRad,Hercules,CA)after ethidium bromide staining.To characterize the cleavability of poly(CBA-DAH-R)under reductive conditions,the polyplexes were prein-cubated with 2.5mM DTT for2h at37°C prior to electrophoresis.
Cells and Cell Culture Conditions.Human prostate carcinoma(PC-3),human oral cavity epidermal carcinoma (KB),human cervical carcinoma(HeLa),human ovarian carcinoma(A2780),and human lung carcinoma(A549)were obtained from American Type Culture Collection(Manassas, VA).Cells were routinely maintained in an RPMI1640 medium supplemented with10%fetal bovine serum.Cells were cultured as a monolayer in a humidi?ed atmosphere containing5%CO2at37°C.Cells were regularly passaged and reseeded24h before transfection experiments.
Cellular Toxicity Assay.Relative cell viability was determined using an MTT(Thiazolyl Blue Tetrazolium Bromide)assay.PC-3cells were plated in96-well plates at a density of1×104cells per well in0.2mL of culture medium and incubated for24h at37°C prior to polyplex treatment.The medium was replaced by a fresh serum-free transfection medium containing a desired amount of the siRNA polyplexes with poly(CBA-DAH),poly(CBA-DAH-R),and bPEI,which were formed at different weight ratios from0:1to60:1.The polyplexes were prepared using0.15μg of siRNA and varying amounts of the polymers in0.1 mL of PBS(pH7.4).Cells were transfected by the polyplexes for4h at37°C and further incubated in a fresh serum-containing medium for24h at37°C.Fifty microliters of MTT solution(2mg/mL)were added and incubated at37°C for4h.The produced formazan crystals were dissolved in0.2mL of DMSO followed by plate reading at530nm in a microplate reader(Bio-Rad680,Hercules,CA).The cell viability was determined relative to the untreated control cells.
Flow Cytometry and Confocal Microscopy.The cellular uptake of the siRNA polyplexes was examined with?ow cytometry and confocal microscopy by using Cy3dye-labeled siRNA.The siRNA polyplexes with poly(CBA-DAH),poly(CBA-DAH-R),and bPEI were used at a weight ratio of40:1,40:1,and1:1,respectively.At the chosen weight ratios of polymer to siRNA,the corresponding calculated charge ratios for poly(CBA-DAH),poly(CBA-DAH-R),and bPEI were43:1,25:1,and8:1,respectively. PC-3cells were plated in6-well plates at an initial density of2.5×105cells per well for?ow cytometry analysis.The siRNA polyplexes were formed with0.75μg of Cy3-siRNA and a desired amount of the polymers in an aqueous buffer solution(PBS0.1mL,pH7.4)for15min at room temperature.The Cy3-siRNA formulations,including naked siRNA,siRNA/poly(CBA-DAH),siRNA/poly(CBA-DAH-R),and siRNA/bPEI polyplexes,were added to the wells containing a serum-free transfection medium.Prior studies in our laboratory had demonstrated that the transfection ef?ciencies for the Cy3-siRNA formulations did not differ between4,8,12and24h of incubation,and so incubation times of4h for the Cy3-siRNA formulations were used throughout the experiments.After4h incubation,cells were washed four times with cold PBS,harvested by trypsin digestion,and?xed in75%EtOH solution for30min at4
articles Kim et al. 720MOLECULAR PHARMACEUTICS VOL.6,NO.3
°C.Cells were analyzed on a?ow cytometer(FACS Caliber, Becton-Dickinson,Mountain View,CA)using FL2channel (Ex.488nm/Em.575nm).Data were processed using Windows Multiple Document interface software(WinMDI). For confocal microscopy,PC-3cells were seeded in confocal imaging dishes(Glass Bottom microwells,MatTek Corp.,Ashland,MA)at a density of1×104cells per well. Cells were preincubated in the presence or absence of10 mM DL-buthionine-sulfoxamine(BSO),which causes a signi?cant decrease in the intracellular level of reduced glutathione(GSH),for24h prior to transfection.The siRNA polyplexes,which were prepared by the same method described above,were transfected for2h at37°C.The transfection medium was replaced with a fresh culture medium and the cells were incubated for a further3h at37°C.The cells were washed with cold PBS four times and ?xed with0.5%para-formaldehyde solution for30min at4°C.The localization of Cy3-labeled siRNA within the cells was visualized by a confocal laser scanning microscope (Olympus Fluoview FV300,Melville,NY)with a100×oil-immersion objective lens using an argon/krypton mixed gas laser(Ex.568nm).Three-dimensional confocal images for the cells were constructed by using Velocity software (Improvision Inc.,Lexington,MA).
In Vitro Transfection.For in vitro transfection studies, 2.5×105cells(PC-3,KB,HeLa,A2780,and A549)per well were plated in a6-well plate.After24h incubation, the culture medium was replaced by the transfection medium with or without10%FBS prior to transfection.To decrease the intracellular reduction potential,cells were preincubated in the presence or absence of10mM BSO at37°C for24h prior to transfection.VEGF siRNA(1.5μg)was condensed with a desired amount of the polymers in0.2mL of PBS. The VEGF siRNA polyplexes were left at an ambient temperature for30min and added into the transfection medium.After4h transfection at37°C,the transfection medium was replaced with a fresh medium containing10% FBS and continuously incubated for a further8h.The medium was removed and supplemented with fresh culture medium containing heparin(20μg/mL).After18h incuba-tion,the medium was collected for VEGF ELISA assay.The amount of VEGF secreted from the cells was determined using a human VEGF immunoassay kit(R&D Systems, Minneapolis,MN)according to the manufacturer’s instruc-tions.All of the data are presented as the mean(standard deviation(SD)of three independent measurements.Statistical analysis was carried out by a Student’s t test.Statistical signi?cance was assigned for p values<0.05.
Results and Discussion
Nonviral vector delivery of synthetic siRNAs can specif-ically silence target genes by inducing mRNA degradation within the cytoplasm.20,21Unlike DNA delivery systems,
which require nuclear localization,synthetic siRNA delivery
systems demand localization to the cytoplasm for RNAi
induction.Localization to the cytoplasm of functional siRNA
molecules is an important step to achieve ef?cient target gene
silencing.Most cationic polymers for gene delivery,however,
condense strongly with nucleic acids and form highly stable
polyplexes in both the extra-and intracellular environment,
even after endosomal escape to the cytoplasm.12Thus,we
hypothesized that bioreducible cationic polymers which
degrade in the reductive environment of the cytoplasm would
be more likely to release siRNAs into the cytoplasm,thereby
resulting in enhanced RNA silencing compared to the
commercially available cationic polymer polyethylenimine,
bPEI(25kDa).
We chose to compare our bioreducible cationic polymer,
arginine-conjugated poly(cystaminebisacrylamide-diamino-
hexane),to bPEI for several reasons.First,bPEI is considered
one of the most successful nonviral gene carriers and is used
extensively for in vitro as well as for in vivo gene delivery
due to its high transfection ef?ciency and reproducibility.
Second,bPEI possesses high endosomal buffering capacity,
which facilitates the endosomal escape of the complexes via
the hypothetical proton sponge effect.Third,while several
cholesterol based cationic lipids have been generated as gene
carriers and have demonstrated high transfection ef?ciency
in mammalian cells,most have lacked an effective endosomal
escape mechanism such as that found in bPEI.For all these
reasons,as well as its wide commercial availability,bPEI is
still accepted as the gold standard for nonviral,polymer based
gene delivery.
siRNA/poly(CBA-DAH-R)Polyplexes Are Degraded under Reductive Conditions.To develop an ef?cient siRNA delivery system,we designed a new bioreducible cationic
polymer poly(CBA-DAH-R)containing multiple disul?de
linkages in the arginine-modi?ed polymeric backbone.Poly-
(CBA-DAH-R)was prepared by arginine modi?cation after
the synthesis of poly(CBA-DAH)(supplemental?gure in the
Supporting Information).The polydisul?de poly(CBA-DAH)
was synthesized from the copolymerization of amine donor
1,6-diaminohexane(DAH)to reducible cystaminebisacryla-
mide(CBA).Then the unprotected terminal amine group in
the backbone of poly(CBA-DAH)was fully modi?ed with L-arginine(R)to produce poly(CBA-DAH-R).The siRNA polyplex formation and the reductive degradation of poly-
(CBA-DAH-R)were assessed by dynamic light scattering
(DLS)measurements(Figure1).Poly(CBA-DAH-R)and
bPEI were complexed with siRNA molecules at200nm in
(20)Kim,H.R.;Kim,I.K.;Bae,K.H.;Lee,S.H.;Lee,Y.;Park,
T.G.Cationic solid lipid nanoparticles reconstituted from low density lipoprotein components for delivery of siRNA.Mol.
Pharmaceutics2008,5,622–631.
(21)Read,M.L.;Singh,S.;Ahmed,Z.;Stevenson,M.;Briggs,S.S.;
Oupicky,D.;Barrett,L.B.;Spice,R.;Kendall,M.;Berry,M.;
Preece,J.A.;Logan,A.;Seymour,L.W.A versatile reducible polycation-based system for ef?cient delivery of a broad range of nucleic acids.Nucleic Acids Res.2005,33,e86.
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diameter with a weight ratio of 40:1and 1:1,respectively.Poly(CBA-DAH-R)started to condense siRNA at a weight ratio of 10:1,though the polyplexes were not stabilized until a weight ratio of 20:1.The siRNA/poly(CBA-DAH-R)polyplexes were stably formed at a few hundred nanometers in size with weight ratios above 20:1.The bioreducible polycation poly(CBA-DAH-R)spontaneously formed stable nanosized polyplexes with siRNA molecules due to the electrostatic interactions between the positively charged side-chain arginine groups of the polymer and the negatively charged phosphate groups of the siRNAs.DLS analysis showed that the particle size of the siRNA/bPEI polyplexes was dramatically increased with an increase in the weight ratio,due to signi?cant particle aggregation of highly positive
charged polyplexes (zeta potential over +35mV).It has been reported that strongly charged particles are rapidly aggregated under physiologic conditions.22To examine whether the siRNA/poly(CBA-DAH-R)polyplexes degrade and release free siRNAs under reductive conditions,the siRNA poly-plexes with poly(CBA-DAH-R)and bPEI were preincubated in 2.5mM DTT solution and the polyplexes were analyzed by DLS.There was no detectable difference in particle size and surface charge for the siRNA/bPEI polyplexes with and
(22)Wang,Y.;Chen,P.;Shen,J.A facile entrapment approach to
construct PEGylated polyplexes for improving stability in physi-ological condition.Colloids Surf.,B 2007,58,188–196
.
Figure 1.Particle sizes and surface charges of siRNA/poly(CBA-DAH-R)and siRNA/bPEI polyplexes.(a)
Hydrodynamic diameters of the polyplexes at various weight ratios.Representative size distribution diagrams and zeta potentials of siRNA/poly(CBA-DAH-R)(b,c)and siRNA/bPEI (d,e)polyplexes at a weight ratio of 40:1and 1:1,respectively.In panels (c,e)and (b,d),the polyplexes were treated with and without 2.5mM DTT,respectively.
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without DTT treatment (Figure 1b,c).In a reductive environment,however,the size distribution peaks for the siRNA/poly(CBA-DAH-R)polyplexes at 200nm particle size disappeared and the zeta potential was almost neutral (-2.7(0.4mV)(Figure 1d,e),suggesting the polyplexes were completely degraded due to the reductive cleavage of the multiple disul?de linkages in the poly(CBA-DAH-R).The unique structural characteristics of poly(CBA-DAH-R)were further con?rmed in the electrophoretic mobility shift assay (Figure 2).Poly(CBA-DAH-R)fully condensed siRNA molecules at weight ratios above 20:1,correlating with the particle formation data determined by DLS analysis (Figure 1).A reductive environment caused complete siRNA release from the siRNA/poly(CBA-DAH-R)polyplexes as a result of degradation of the polymers,while the structure of the siRNA/bPEI polyplexes was not affected by a reductive environment.Previous studies have shown that polydisul?de polycations exhibit ef?cient unpackaging of the polyplexes by the cleavage of disul?de bonds in reductive conditions,which is responsible for the release of nucleic acids.16-18The triggered degradation behavior of the siRNA/poly(CBA-DAH-R)polyplexes increases RNA silencing ef?ciency by improving the localization of the siRNA to the cytoplasm.
siRNA/poly(CBA-DAH-R)Polyplexes Decrease Cyto-toxicity and Enhance Transfection Ef?ciency.To assess the ef?cacy of poly(CBA-DAH-R)as an siRNA carrier in vitro,the cytotoxic activity of the siRNA/poly(CBA-DAH-R)polyplexes against PC-3cells was evaluated by MTT assay (Figure 3a).The siRNA/poly(CBA-DAH-R)poly-plexes had much lower cytotoxicity than the bPEI formula-tions.Poly(CBA-DAH-R)exhibited nearly 100%relative cell viability at a weight ratio up to 40:1,while bPEI showed only 40%relative cell viability at a weight ratio near 10:1.There was no meaningful difference in cellular toxicity between the unmodi?ed and the arginine-modi?ed reducible polymers,suggesting that the arginine modi?cation does not affect the biocompatibility of bioreducible cationic polymers.The cytotoxicity of polycations is caused by an increase in molecular weight as well as cationic charge density.23The cytotoxicity of higher molecular weight polycations,how-ever,decreases with breakdown into lower molecular weight components.24Thus,the improved cell viability of poly-(CBA-DAH-R)may be attributable to its biodegradability.Further in vitro studies conducted at nontoxic levels of bPEI,poly(CBA-DAH),and poly(CBA-DAH-R)formulations dis-played over 90%cell viability with weight ratios under 1:1,40:1,and 40:1,respectively.
To investigate the in?uence of the arginine modi?cation of poly(CBA-DAH-R)on transfection ef?ciency,the cellular uptake of ?uorescently labeled siRNA formulations by PC-3cells was monitored using ?ow cytometric analysis (Figure 3b).Most of the control and the naked siRNA treatment groups showed very low ?uorescence intensity.However,the ?uorescence peaks for the cells treated with various polyplex formulations were notably shifted to the right.The percentage of the cells gated from region M for the siRNA/poly(CBA-DAH),siRNA/poly(CBA-DAH-R),and siRNA/bPEI polyplexes was 57.0%,81.0%and 88.2%,respectively.The extent of cellular uptake of the siRNA/poly(CBA-DAH-R)polyplexes was 1.4-fold greater than that of the siRNA/poly(CBA-DAH)polyplexes.There was no signi?cant difference,however,between poly(CBA-DAH-R)and bPEI in the cellular uptake of the polyplexes.The enhanced delivery of the siRNA/poly(CBA-DAH-R)polyplexes is likely due to the arginine modi?cation of the polydisul?de cationic polymers.Arginine residues,which are rich in membrane translocalization peptides,help enhance the cel-lular association and membrane permeability of many biologically active products.19
To assess the potential ef?cacy of siRNA therapy for cancer,the RNAi activity of the siRNA/poly(CBA-DAH-R)polyplexes was evaluated in human prostate carcinoma PC-3cells using siRNA targeted against VEGF.VEGF has a predominant role in tumor angiogenesis (Figure 3c).25,26The VEGF siRNA polyplex formulations silenced VEGF gene expression in a sequence speci?c manner.Both the
(23)Fischer,D.;Li,Y.;Ahlemeyer,B.;Krieglstein,J.;Kissel,T.In
vitro cytotoxicity testing of polycations:in?uence of polymer structure on cell viability and hemolysis.Biomaterials 2003,24,1121–1131.
(24)Kim,Y.H.;Park,J.H.;Lee,M.;Kim,Y.H.;Park,T.G.;Kim,
S.W.Polyethylenimine with acid-labile linkages as a biodegrad-able gene carrier.J.Controlled Release 2005,103,209–219.(25)Kim,S.H.;Jeong,J.H.;Lee,S.H.;Kim,S.W.;Park,T.G.
Local and systemic delivery of VEGF siRNA using polyelectrolyte complex micelles for effective treatment of cancer.J.Controlled Release 2008,129,107–116.
(26)Hicklin,D.J.;Ellis,L.M.Role of the vascular endothelial growth
factor pathway in tumor growth and angiogenesis.J.Clin.Oncol.2005,23,1011–1027
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Figure 2.Gel retardation assay of siRNA/poly(CBA-DAH-R)polyplexes at various weight ratios without DTT treatment
(a)and with 2.5mM DTT treatment (b).siRNA/bPEI polyplexes were formulated at a weight ratio of 1:1.
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siRNA polyplexes with poly(CBA-DAH)and poly(CBA-DAH-R)decreased VEGF expression.As expected,the arginine-containing polyplexes showed consistently greater gene silencing ef?ciency than the unmodi?ed polyplexes,presumably resulting from the enhanced cellular uptake of the siRNA/poly(CBA-DAH-R)polyplexes.The VEGF siRNA/poly(CBA-DAH-R)polyplexes formulated at a weight ratio of 40:1exhibited prominent VEGF silencing activity,compared to the VEGF siRNA/poly(CBA-DAH)and VEGF siRNA/bPEI (a weight ratio of 1:1)polyplexes.Meanwhile,the VEGF siRNA/poly(CBA-DAH)polyplexes demonstrated a similar level of VEGF silencing as the bPEI formulations,though the siRNA/poly(CBA-DAH)polyplexes showed a lower level of cellular association than the siRNA/bPEI polyplexes (Figure 3b).These results imply that high-ef?ciency uptake of siRNA polyplexes does not necessarily guarantee the RNAi activity of siRNA therapeutics.In addition to enhancing the cellular delivery with arginine modi?cation,the unique structural characteristic of poly-(CBA-DAH-R)appears to exert considerably more in?uence in siRNA-mediated gene silencing compared to other poly-mer carriers.
Localization to the Cytoplasm of siRNA Molecules by Poly(CBA-DAH-R)Polyplexes Enables Strong RN-Ai Induction.To verify the hypothesis that reductive degradation of the siRNA/poly(CBA-DAH-R)polyplexes leads to improved RNA silencing,confocal microscopic observations on subcellular localization of ?uorescein binding siRNA were carried out using PC-3cells (Figure 4).In the cells transfected by the siRNA/poly(CBA-DAH-R)poly-plexes,siRNA was evenly distributed throughout the cyto-plasm.However,the cytoplasmic localization of siRNA was fully suppressed with the treatment of a glutathione depleting agent,BSO,which is known to inhibit intracellular reducing potential.27These ?ndings demonstrate that intact siRNA molecules were successfully released from the destabilized polyplexes due to the degradation of the polymer backbones in the reductive environment of the cytoplasm.In contrast,small ?uorescent spots were observed throughout the cells when transfected with the siRNA/bPEI polyplexes,irrespec-tive of the presence or absence of BSO,suggesting that most of the siRNA molecules were still complexed in the large bPEI polyplex aggregates due to the limited degradation of the bPEI polyplexes and release of siRNA at the cellular level.These observations indicate that the triggered release of siRNA from the siRNA/poly(CBA-DAH-R)polyplexes could play a critical role in enhancing RNA silencing.To further con?rm this hypothesis,the VEGF gene silencing experiments in the PC-3cells were conducted under both reducing and nonreducing conditions (Figure 5a).When the VEGF siRNA/poly(CBA-DAH-R)polyplexes were trans-fected in the presence of 10mM BSO,VEGF expression was signi?cantly higher,while the bPEI polyplexes exhibited
(27)Shalini,S.;Bansal,M.P.Co-operative effect of glutathione
depletion and selenium induced oxidative stress on API and NFkB expression in testicular cells in vitro:insights to regulation of spermatogenesis.Biol.Res.2007,40,307–317
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Figure 3.(a)Cellular toxicity assay of poly(CBA-DAH),
poly(CBA-DAH-R),and bPEI polyplexes with siRNA as a function of weight ratio.(b)Representative ?ow cytometry histograms of PC-3cells transfected with naked siRNA (green),siRNA/poly(CBA-DAH)(yellow),siRNA/poly(CBA-DAH-R)(red),and siRNA/bPEI (blue).Cy3-modi?ed siRNA was used.M presents a gated region (?uorescence intensity (arbitrary unit):350-10,000).(c)VEGF gene silencing of VEGF siRNA/poly(CBA-DAH-R)polyplexes at various weight ratios.PC-3cells were transfected with bPEI (weight ratio 1:1),poly(CBA-DAH),and poly(CBA-DAH-R)formulations with VEGF siRNA.GFP siRNA/bPEI polyplexes were used as a control.Statistical signi?cance,*)p <0.05versus bPEI,**)p <0.05versus poly(CBA-DAH).The VEGF expression was analyzed using an ELISA for human VEGF.
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the same level of RNAi activity against VEGF expression in both BSO treated and BSO untreated cells.These results indicate that the biodegradability of poly(CBA-DAH-R)increases the localization of siRNA to the cytoplasm.
VEGF siRNA/poly(CBA-DAH-R)Polyplex Delivery System for the Treatment of Various Human Carci-nomas.The in?uence of serum proteins upon the VEGF gene silencing activity of the VEGF siRNA/poly(CBA-DAH-R)polyplexes was evaluated by conducting the transfection under 10%FBS conditions (Figure 5b).Although both poly(CBA-DAH-R)and bPEI showed approximately a 2-fold reduction in RNAi activity in the presence of serum proteins,the VEGF siRNA/poly(CBA-DAH-R)polyplexes still ex-hibited a higher level of VEGF inhibition compared to the VEGF siRNA/bPEI polyplexes.These results suggest that the VEGF siRNA/poly(CBA-DAH-R)polyplex delivery system could be a potential approach for siRNA-based cancer therapy.
To make the VEGF siRNA/poly(CBA-DAH-R)formula-tion applicable to a wide range of tumor types,the VEGF gene silencing activity of the VEGF siRNA/poly(CBA-DAH-R)polyplexes was further assessed in various human cancer cells including oral (KB),cervical (HeLa),ovarian (A2780),and lung (A549)in addition to the prostate carcinoma PC-3cells (Figure 6).The cancer cell lines manifested different levels of VEGF production and VEGF inhibition by the
VEGF siRNA formulations.It is known that cancer cells upregulate VEGF expression,but the degree of upregulation varies,depending on the cancer cell type.26,28The differences in VEGF gene silencing of the VEGF siRNA/poly(CBA-DAH-R)polyplexes in varied cancer cell types are probably determined by differences in the levels of intracellular reducing potential,since different cells have different capaci-ties for glutathione synthesis to maintain the reductive environment of the cytoplasm.29Poly(CBA-DAH-R)invari-(28)Roberts,W.G.;Delaat,J.;Nagane,M.;Huang,S.;Cavenee,
W.K.;Palade,G.E.Host microvasculature in?uence on tumor vascular morphology and endothelial gene expression.Am.J.Pathol.1998,153,1239–1248.
(29)Wang,W.;Ballatori,N.Endogenous glutathione conjugates:
occurrence and biological functions.Pharmacol.Re V .1998,50,335–356
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Figure 4.Subcellular confocal images of cytoplasmic
siRNA release of siRNA/poly(CBA-DAH-R)and siRNA/bPEI polyplexes with and without glutathione-depleting agent BSO.Cy3-modi?ed siRNA was used.Scale bars represent 10μ
m.
Figure 5.(a)Effect of glutathione-depleting agent BSO
on VEGF gene silencing of VEGF siRNA/poly-(CBA-DAH-R)polyplexes in PC-3cells.Cells were pretreated with or without 10mM BSO for 24h prior to transfection.Statistical signi?cance,*)p <0.01versus w/o 10mM BSO.(b)Effect of serum proteins on VEGF gene silencing of VEGF siRNA/poly(CBA-DAH-R)polyplexes in PC-3cells.Cells were transfected in the media with or without 10%FBS.Statistical signi?cance,*)p <0.05versus bPEI (w/o 10%FBS),**)p <0.01versus bPEI (w/10%FBS).The amount of VEGF secreted from the cells was determined using an ELISA for human VEGF.
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ably exhibited higher RNAi activity than bPEI in all cancer cell lines tested,suggesting that the VEGF siRNA/poly(CBA-
DAH-R)polyplex delivery system could be useful in the treatment of various types of human cancers.To validate the current in vitro study,we are currently conducting an in vivo study using a human tumor xenograft model,which we anticipate reporting separately in the near future.In conclusion,we have demonstrated a new approach for RNAi gene silencing using a VEGF siRNA/poly(CBA-DAH-R)polyplex delivery system.The siRNA/poly(CBA-DAH-R)polyplexes successfully localize siRNA to the cytoplasm due to the reductive degradation of the polymers.In addition to the feature of triggered release,arginine modi?cation of poly(CBA-DAH-R)enhances cellular permeability,leading to effective down regulation of VEGF expression in various human cancer cells.Since the siRNA/poly(CBA-DAH-R)polyplex formulation has low cytotoxicity,high ef?ciency in target gene silencing,and broad ef?cacy in various cell types,the siRNA/poly(CBA-DAH-R)polyplex offers a broad range of potential applications for delivering therapeutic siRNAs,including cancer,obesity,heart disease,and diabetes.Acknowledgment.This work was supported by grants from the National Institutes of Health HL071541(D.A.B.)and CA107070(S.W.K.)and the Korea Research Foundation (KRF-2007-357-D00074).
Supporting Information Available:Description of supple-mentary experimental methods and ?gures.This material is available free of charge via the Internet at https://www.doczj.com/doc/b113130527.html,.
MP800161E
Figure 6.VEGF gene silencing of VEGF siRNA/
poly(CBA-DAH-R)polyplexes in various human cancer cells.Statistically signi?cant difference from bPEI,*)p <0.05,**)p <0.01.The VEGF expression level was determined using an ELISA for human VEGF.
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siRNA细胞转染实验操作指南 siRNA产品的最佳工作浓度、转染后检测时间因不同的细胞类型和研究目的而异。一般推荐的siRNA工作浓度为50nM，检测时间为转染后24~72h。可以通过设置时间梯度和浓度梯度进行组合实验来选择最优的siRNA工作浓度和检测时间。由于siRNA过量会对细胞产生毒性，实验建议的siRNA 工作浓度优化的范围为5~100nM。 下面以Lipofectamine 2000转染24孔板的293T细胞为例，选取50nM的siRNA工作浓度，介绍siRNA转染细胞的操作步骤。若使用其他的转染试剂，请参照对应的产品说明书进行操作。 1.在转染前一天，按1×105~5×105个/孔的量将细胞接种于不含抗生素的完全培养基中，使次日转染时细胞融合度30-50%为宜。接种时尽量 保证每孔细胞的接种数量一致，使细胞均匀地平铺在生长表面。 注意:降低转染时的细胞密度可延长转染和收样的时间间隔，避免细胞过度生长对实验结果造成影响。可根据细胞特性和实验目的等，调整接种时的细胞密度。 2.每个待转染的细胞样品（每个培养孔），按以下体系配置转染所需的siRNA-Lipofectamine 2000复合物: （1）配制稀释液A:取25pmol siRNA于50ul Opti-MEM无血清培养基中稀释，轻轻混匀。（*siRNA的用量计算参照表1） （2）配制稀释液B:使用前先将Lipofectamine 2000轻轻混匀，取出1ul于50ul Opti-MEM无血清培养基中稀释，轻轻混匀，室温下孵育5分钟。注意孵育时间不能超过25min。 （3）孵育完成后，将稀释液A与B轻轻混匀得到siRNA-Lipofectamine 2000复合物，在室温下孵育20分钟。此时溶液可能会浑浊，属于正常现象。 表1 不同细胞培养板siRNA 转染用量参考表 3.将孵育好的siRNA-Lipofectamine 2000复合物分别加到对应的细胞孔中，轻轻混匀。 注意：转染前无需更换新鲜培养基，直接将复合物加到原细胞培养基中即可。如有需要，也可根据情况更换培养基优化该步骤。 4.如果需要进行其他特殊处理，如加药等，可在此时进行。 5.将细胞培养板置于37℃的CO2培养箱中培养24-72h。具体培养时间根据细胞生长特性、实验目的进行调整。 注意：加入siRNA-Lipofectamine 2000复合物后一般无需更换培养基，但若脂质体类转染试剂对细胞毒性较大，可根据细胞的状态，在转染4-6小时后更换培养基。 6.进行后续总RNA提取、蛋白提取、细胞活性分析等实验。
FuGENE HD真核细胞转染流程 FuGENE HD Transfection Reagent Quick Protocol.pdf FuGENE HD Transfection Reagent.pdf 1.细胞和质粒的准备 ①将约5-10×105A549细胞接种于6孔细胞板中，用含10%小牛血清的F-12k 培养过夜，约60%~80%铺满时用于转染。 注：一般荧光试验细胞稀一点为宜；WB试验细胞密一点为宜 ②转染时所用质粒均用转染专用质粒抽提试剂盒无菌提取，抽提后测定其浓度 和纯度，浓度在0.1μg/μL以上且纯度在1.8±0.5 (OD260/OD280)的质粒用于转染，质粒提取的具体步骤按说明书提供的方法进行。（注：质粒浓度测定未必可行，需同时进行酶切和PCR鉴定方可。） ③按照转染剂量，计算质粒使用浓度 2.转染流程 注：由于不同转染试剂对质粒大小、性质、转染量、细胞的种类有很强的特异性，必须谨慎选择转染试剂。以多次转染成功使用的转染试剂为佳。 ①将培养板中的上清弃去，用细胞用无抗无血清F12K洗涤三次后，每孔加入 800ul 无抗无血清F12K。 ②取出FuGENE HD转染试剂，使其温度上升到室温。 ③计算好质粒、转染试剂、以及无抗无血清F12K的量，最终液体总量为100 ul。 准备好无菌指形管，按计算结果加入90-98ul的无抗无血清F12K，加入2ug 质粒，振荡器混匀；随后加入6ul FuGENE HD转染试剂，立即震荡混匀（转染试剂加入时不能沾到管壁上！） ④室温静置7-12分钟后，将质粒：转染试剂混合液均匀滴加在细胞平板孔中， 吹打混匀或者震荡混匀。放入37℃培养箱孵育。
产品以冻干粉的形式，常温运输。收到产品后，请于-20 C?-80 'C保存，冻干粉可以稳定保存一年。 使用前请瞬时离心，用RNase-freeH 2O或者灭菌ddH20，配制成20gM储存液，分装保存，避免反复冻 融（尽量不超过5次）。 表120 gM储存液的配置参考 使用前须知 为避免外界因素（包括酶，极端pH或者温度条件等）导致产品降解，所有操作请严格遵循RNA操作规则。 实验过程中，产品最好于冰上放置，使用完毕后请于-20 C~-80 C小心保存。 细胞实验方法 为了降低细胞密度、试剂用量，转染效率等因素导致的孔间差异，保证实验可靠性和可重复性，一般建议： a. 转染实验中每个转染样品至少设置3个以上复孔； b. 接种细胞时，每孔接种的细胞数量尽量保持一致，并且细胞在各孔的表面平均分布。 1. 转染浓度： siRNA产品最佳工作浓度因不同的细胞类型及研究目的而异。推荐初始转染浓度为50nM，转染后检测时 间为24?72h。最佳转染效率一般通过设置时间曲线和浓度梯度进行优化，优化的范围建议为5~100nM。 2. 转染步骤： 以Iipofectamine2000（简称Iipo2000）转染siRNA于24孔板，转染浓度为50nM为例，其他规格容器转染 请参考表2。 1）转染前一天，接种适当数量的细胞至细胞培养板中，每孔中加入不含抗生素的培养基，使转染时的细胞 密度能够达到30~50%（不同细胞生长速度不一样，因此，接种细胞的数量需要根据细胞培养的经验）。注意: 转染时，细胞密度是影响转染效率的关键因素之一，细胞生长过度会削弱细胞活力，降低细胞的转染效率。 2）对于每个转染样品，请按以下步骤准备siRNA-Iipo2000混合液： a. 稀释siRNA :用50 g 不含血清培养基Opti-MEM I （v1）稀释g I20 gM siRNA储存液，轻轻混匀，室温孵育5min ; b. 稀释Iipo2000 :用50 g不含血清培养基Opti-MEM I （v1）稀释1 g Ilipo2000，轻轻混匀并室温孵育5min ; c. 将a与b轻轻混匀，室温孵育20min（溶液可能会有浑浊，但不会影响转染）。 注意：稀释好的Iipo2000长时间放置可能导致转染试剂活性的降低，应尽量在25min之内与稀释好的siRNA 混合。另在混合试剂时，请勿剧烈吹打或振荡，手指轻弹管壁即可，过度用力可能会破坏脂质体的结构， 甚至影响siRNA-lipo2000 混合物的形成。 3）将siRNA-Iipo2000混合液加入含有细胞的400 gl培养基（v2）的培养孔中，轻轻混匀； 4）（可选）培养4?6h后，将孔中含siRNA-Iipo2000混合液的培养基移去，更换新鲜培养基； 5）（可选）进行其他必要的特殊处理（如加药处理）；
Stealth? RNAi or siRNA Transfection 以24孔板为例，其余规格的转染见表1 1 中板，细胞密度为30-50%适宜。 注意：根据转染后细胞检测时间长短决定细胞中板密度，如果转染后需要长时间后检测，则细胞中板密度适当降低，已避免细胞过度生长导致存活降低。 2 第二天（24-36小时后）每个孔转染方式如下： A 将20pmol siRNA溶于50ul Opti-mem无血清培养基中。 B 将1ul lipo2000溶于50ul Opti-mem无血清培养基中，混匀室温放置5min。 C 将A B两管混合，放置20min。 3 转染期间，将24孔板培养基换成无血清培养基，每孔400ul。将C管mix加入24孔板对应孔中，4-6小时候换成有血清培养基。 Plasmid DNA Transfection DNA(ug):lipo 2000(ul)=1:2-3 转染时细胞密度越高，转染效率，表达效率也越高，并且可以降低细胞毒性。 1 中板。 贴壁细胞：0.5-2X105 cells/well，第二天待细胞密度达到90%以上时转染 悬浮细胞：4-8X105 cells/well，中板后随即转染。 2 转染。 A 将0.8ug DNA溶于50ul Opti-mem无血清培养基中。 B 将2ul lipo2000溶于50ul Opti-mem无血清培养基中，混匀室温放置5min。 C 将A B两管混合，放置20min。 转染期间，将24孔板培养基换成无血清培养基，每孔400ul。将C管mix加入24孔板对应孔中，4-6小时候换成有血清培养基。
Table 1. Culture Shared reagents DNA transfection RNAi transfection *：中板密度根据不同细胞不同实验有所不同，这里仅提的数据仅供参考 **：6孔板细胞质粒转染量1-2ug足以。 ***：6cm dish细胞质粒转染量4-6ug足以。
siRNA使用说明（2009-08） 名称： 常规化学合成siRNA。 产品简介： 常规化学合成siRNA为21-25nt，带有2nt的3'端悬垂的双链小RNA（3'端悬垂通常为dTdT或者UU，也可以选择其他的碱基作为3'端悬垂）。 产品剂量经过严格测算，以摩尔数标明。产品剂型为冻干粉，即用型的siRNA已经经过去保护、退火、纯化等处理，只要用灭菌的ddH2O或者RNase-free water溶解并配制成20μM液体即可直接用于细胞转染及其他实验。 保存和使用： 保存： 液体剂型，siRNA的贮存浓度一般为20μM，-20℃或-70℃保存半年以上； 冻干粉剂型，-20℃或-70℃保存一年以上。开盖前，请先稍稍离心，将粉末收集管底，再用灭菌的ddH2O或者RNase-free water，配制成20μM的液体剂型。 例如：20nmolsiRNA冻干粉，需要加入1ml水溶解（其他剂量可以按照比例计算加水量）。 注意：（1）液体剂型产品，应避免反复冻融（尽量不要超过5次），建议溶解后的产品分装保存。（2）如果近期不做实验，产品需要长期放置，最好以冻干粉形式保存。 使用： 产品使用时（转染过程），siRNA最好冰上放置，使用完毕，请于-20℃或-70℃小心保存; 整个实验过程，要求无RNA酶环境，枪头、EP管都要经DEPC处理； 特别提示：荧光标记siRNA要求避光。 使用方法： 1. siRNA的转染浓度：推荐的siRNA转染浓度是50nM，客户可根据实验具体情况优化转染浓度，优化的范围可以是10～150nM。 （siRNA及转染试剂的建议用量，请参照“实验指导”）。 2. 使用lipofectamine2000（Invitrogen）转染siRNA的步骤（仅供参考）： (1) 转染前一天，接种适当数量的细胞至细胞培养板中，使转染时的细胞密度能够达到30～50％（不同细胞生长速度不一样， 因此，接种细胞的数量需要根据细胞培养的经验），请使用无抗生素的培养基。 注意：转染时，细胞密度是影响转染效率的关键因素之一，细胞生长过度会削弱细胞活力，从而降低细胞的转染效率。而细胞密度过低则可能达不到生长的要求，也会因此影响转染效率。 (2)对于每个转染样品，按如下步骤准备siRNA- lipo2000混和液（试剂的用量和体积，请参照“实验指导”）： a. 稀释转染试剂lipofectamine2000（以下称lipo2000）：使用前，将lipo2000转染试剂轻轻摇匀，然后取适量，用不含血清 的优化培养基（Opti- MEMⅠ）稀释，轻轻混和，室温孵育5min； b. 稀释siRNA：用不含血清的Opti -MEMⅠ稀释siRNA，轻轻混和； c. 稀释好的lipo2000经过5min的孵育后，将之与上述（b）稀释好的siRNA轻轻混和，室温培养20min以形成siRNA-lipo2000 混和物，溶液可能会有浑浊，不过不会影响转染。 注意：稀释好的lipo2000，如果长时间放置可能导致转染试剂活性的降低，应尽量在30min之内与稀释好的siRNA混和。 (3) 将siRNA-lipo2000混和液加入含有细胞以及培养液的细胞培养板中，轻轻摇晃，使之混和； (4)将培养板置于37 ℃的CO2培养箱中培养至检测时间（24～96h）。沉默效率的检测一般建议的时间为24～72h。 可选操作（并非必要的操作）：转染操作完成，经过37℃培养4～6h后，可以将孔里含有siRNA-lipo2000混合液的培养基移去，更换新鲜的生长培养基，这样也不会影响转染的效率。 注意：如果转染时使用的是不含血清的培养基（即血清饥饿的条件下进行转染），4～6h后必须换成完全培养基（含血清、含抗生素），以确保细胞生长。 3. mRNA 水平的检测：siRNA的作用机理在于其引起靶mRNA的降解，因此mRNA的降解水平是siRNA沉默效率的最直接指标。 一般在siRNA转染后24h即可以检测到靶mRNA水平的降低。检测方法一般采用RT-PCR（半定量）或Real-time PCR（定量）。 4. 蛋白水平的检测：蛋白水平的检测一般需要借助抗体（针对靶基因蛋白质的抗体或针对融合蛋白的抗体）或报告基因系统检 测，检测手段一般有Western-blot、免疫组化等。一般说来，检测时间受细胞内蛋白质表达量、蛋白质本身的半衰期等因素的影响，一般从48h开始检测，在36、48、72、96h，甚至更长时间之后多点采样。
THE RNAi COMPANY RNAi 产品使用手册 上海吉玛制药技术有限公司 Shanghai GenePharma Co.,Ltd.
Ⅰ. RNAi 简介 1 A. RNAi 实验原理 B. RNAi 实验流程 C. RNAi 实验所需试剂 D. 上海吉玛 RNAi 相关产品 Ⅱ. siRNA设计7 A. 哺乳动物siRNA设计 B. 上海吉玛 siRNA 产品特性 C. siRNA oligo 技术数据 Ⅲ. siRNA 对照9 A. 普通阴性对照 B. 荧光标记的阴性对照 C. siRNA阳性对照 D. 转染试剂对照 E. 避免off-target对照 Ⅳ. siRNA 转染10 A.siRNA 转染的方法 B.Lipofectamin2000 转染试剂 C.Lipofectamin2000适用的细胞类型 D.转染前细胞培养 E.Lipofectamin：siRNA/DNA比例 F.贴壁细胞转染程序 G.悬浮细胞siRNA转染程序 H.DNA和siRNA共转染细胞程序 I. 体内siRNA导入方法 J. siRNA转染常见问题与建议 Ⅴ. mRNA水平RNAi效果监测15 A. siRNA细胞转染条件优化 B. Real-Time PCR RNAi 效果检测 C. Real-Time PCR 结果分析 Ⅵ. 蛋白质水平RNAi效果监测20 A. western-blot原理 B.western-blot操作步骤 w C.estern-blot上样液的制备 D.western-blot常用试剂的配制 Ⅶ. RNAi实验常见问题解答22
Ⅰ. RNAi 简介 A. RNAi实验原理 RNA干扰（RNA interfering，RNAi）现象是由与靶基因序列同源的双链RNA（double-stranded RNA，dsRNA）引发的广泛存在于生物体内的序列特异性基因转录后的沉默过程。细胞中的核糖核酸酶III家族成员之一的，dsRNA特异性的核酸酶Dicer将dsRNA裂解成由21-25个核苷酸组成的小干扰RNA (small interfering RNA，siRNA)，随后siRNA作为介导子引起特异性地降解相同序列的mRNA，从而阻断相应基因表达的转录后基因沉默机制。
细胞转染技术原理及应用 常规转染技术可分为两大类，一类是瞬时转染，一类是稳定转染（永久转染）。前者外源DNA/RNA不整合到宿主染色体中，因此一个宿主细胞中可存在多个拷贝数，产生高水平的表达，但通常只持续几天，多用于启动子和其它调控元件的分析。一般来说，超螺旋质粒DNA转染效率较高，在转染后24-72小时内（依赖于各种不同的构建）分析结果，常常用到一些报告系统如荧光蛋白，β半乳糖苷酶等来帮助检测。后者也称稳定转染，外源DNA 既可以整合到宿主染色体中，也可能作为一种游离体（episome）存在。尽管线性DNA比超螺旋DNA转入量低但整合率高。外源DNA整合到染色体中概率很小，大约1/104转染细胞能整合，通常需要通过一些选择性标记，如来氨丙基转移酶（APH；新霉素抗性基因），潮霉素B磷酸转移酶（HPH），胸苷激酶（TK）等反复筛选，得到稳定转染的同源细胞系。 转染技术的选择对转染结果影响也很大，许多转染方法需要优化DNA与转染试剂比例，细胞数量，培养及检测时间等。一些传统的转染技术，如DEAE右旋糖苷法，磷酸钙法，电穿 孔法，脂质体法各有利弊 近年来国际上推出了一些阳离子聚合物基因转染技术，以其适用宿主范围广，操作简便，对细胞毒性小，转染效率高受到研究者们的青睐。其中树枝状聚合物（Dendrimers）和聚乙烯亚胺（Polyethylenimine，PEI）的转染性能最佳，但树枝状聚合物的结构不易于进一步改性，且其合成工艺复杂。聚乙烯亚胺是一种具有较高的阳离子电荷密度的有机大分子，每相隔二个碳个原子，即每“第三个原子都是质子化的氨基氮原子，使得聚合物网络在任何pH 下都能充当有效的“质子海绵”(proton sponge)体。这种聚阳离子能将各种报告基因转入各种种属细胞，其效果好于脂质聚酰胺，经进一步的改性后，其转染性能好于树枝状聚合物，而且它的细胞毒性低。大量实验证明，PEI是非常有希望的基因治疗载体。目前在设计更复杂 的基因载体时，PEI经常做为核心组成成分。 线型PEI（Line PEI,LPEI）与其衍生物用作基因转染载体的研究比分枝状PEI（Branched PEI,BPEI）要早一些，过去的研究认为在不考虑具体条件，LPEI/DNA转染复合物的细胞毒性较低，有利于细胞定位，因此与BPEI相比应该转染效率高一些。但最近研究表明BPEI 的分枝度高有利于形成小的转染复合物，从而提高转染效率，但同时细胞毒性也增大。超高分枝的、较柔性的PEI衍生物含有额外的仲胺基和叔胺基，在染实验中发现这种PEI的毒性 低，但转染效率却较高。 GenEscort是采用各种分枝状和超高分枝状的小分子PEI与各种含有生理条件下可降解键的交联剂交联，合成出的一系列高分枝的可降解的PEI衍生物。聚合物的分枝结构使得其具有较高的正电性，因此易于高效地包裹各种DNA、RNA分子及质粒形成小的纳米颗粒，从而提高转染效率，当所形成复合物进入细胞以后，其中所含的生理条件下可降解的化学键在细胞内水解，使交联聚合物分解为无细胞毒性的小分子PEI，这样结构的转染试剂在体外应用可以获得高的转染效率和低的细胞毒性，其可降解性对体内应用也具有重要的意义。
Polyplus-transfection S.A. - Bioparc - 850 Bd S. Brant - 67400 Illkirch - France - Phone: +33 3 90 40 61 80 - Fax: +33 3 90 40 61 81 Polyplus-transfection Inc. - 1251 Ave of the Americas - 34th fl. - New-York - NY 10020 - USA https://www.doczj.com/doc/b113130527.html, jetPRIME? in vitro DNA & siRNA transfection reagent PROTOCOL DESCRIPTION jetPRIME? is a novel powerful molecule based on a polymer formulation manufactured at Polyplus-transfection?. jetPRIME? ensures effective and reproducible DNA and siRNA transfection into mammalian cells. jetPRIME? is extremely efficient on a wide variety of cell lines. This powerful reagent only requires low amounts of nucleic acid per transfection, hence resulting in very low cytotoxicity . 1 Transient DNA transfection protocol (2) 1.1 Cell seeding .......................................................................................................................................... 2 1.2 DNA transfection protocol ................................................................................................................... 2 1.3 Virus production in adherent cells ....................................................................................................... 5 1.4 Optimization guidelines (5) 2 siRNA transfection protocol (6) 2.1 Cell seeding .......................................................................................................................................... 6 2.2 siRNA transfection protocol .. (7) 3 DNA & siRNA cotransfection protocol (7) 3.1 Cell seeding .......................................................................................................................................... 7 3.2 DNA & siRNA cotransfection protocol . (8) 4 Transfection of CRISPR/Cas9 ..................................................................................... 9 5 Stable DNA transfection ............................................................................................. 9 6 Troubleshooting ....................................................................................................... 10 7 Product information (11)
siRNA转染常见问题解答 SiRNA导入细胞有以下几种方法：化学转染技术、电穿孔法、磷酸钙共沉淀技术、显微注射和载体导入技术。选择时应该依据实验条件考虑以下因素：细胞对转入方式的承受能力、细胞对病毒侵染的易感性、细胞的生长特性等。其中，化学转染技术是目前最为常用的方法，由于电转的方法对细胞损伤比较大，一般不建议选择电转。 针对最常见的化学转染技术，有几种常见的问题以及解决方法。 一、哪种转染试剂效果好？ 在选择转染试剂时，一般要考虑的是结合特定的细胞株，而不是被导入细胞中的物质。选择细胞毒性小，转染效率高的转染试剂。脂质体试剂的毒性较大，建议选择非脂质体的转染试剂，如BIODAI的RFect系列纳米材料转染试剂。 二、转染后出现细胞死亡是什么原因？如何优化转染条件？ 转染后细胞死亡，原因也是多样的，如脂质体毒性，转染浓度过高，转染前的细胞状态不佳等都可能导致转染后细胞死亡的情况发生，这种情况下就需要适当优化转染条件；在优化转染条件时需要考虑以下因素：转染试剂和细胞特有的自身条件。例如：siRNA 与转染试剂的比例、转染时间、细胞传代数和细胞密度等。一般说，转染试剂毒性小，转染时所需的细胞密度就小，如RFect系列siRNA转染试剂一般要求30-50%细胞密度，而lipo2000转染时所需的细胞密度一般在70%左右。如果经优化后细胞死亡仍很多，应及时考虑更换转染试剂。 三、如何优化siRNA与转染试剂的比例？ SiRNA的量与转染试剂的比例需要进行优化，一般选择24孔板进行优化，比 较节省各种试剂。可以在10-100nM之间设定几个siRNA的浓度水平，如30nM， 50nM，80nM，100nM。转染试剂根据说明书推荐的剂量上下浮动各3个浓度。之后siRNA的量与转染试剂的量进行两两组合，从中选择转染效率最高的组合用于接下 来的实验。如果通过荧光显微镜观察荧光判断转染效率的话，siRNA的最低终浓度 不要低于10nM，一般推荐的siRNA终浓度多在50-100nM。这里需要说明的是，以
细胞转染的详细过程 1、准备工作如下： 1)从pFastBacTM construct中纯化重组的bacmind DNA(500ng/μl溶于TE中) 从相应的pFastBacTM construct对照中纯化bacmind DNA(500ng/μl溶于TE中)。 细胞培养在适合的培养基中。 细胞转染剂Cellfectin（4℃储存）无任何添加物（例如：FBS，抗生素等）的细胞培养基。用于细胞培养的完全生长培养基（例如：Sf-900ⅡSFM TNMFH 或其他适合的培养基）。2)在6孔板或是35毫米dish上，每孔培养9*105Sf9细胞，细胞培养于2ml含抗生素的生长培养基中。 3)细胞在27℃孵育至少一小时。 4)对于每一个转染的样品，准备bacmid DNA：与细胞转染剂在12*75mm消毒管中进行如下混合： 用100μl无血清培养基稀释1μl纯化的bacmid DNA。 用100μl无血清培养基稀释6μl 细胞转染剂。 将bacmind DNA与细胞转染剂进行混合，动作要轻柔，混合物在室温下孵育45分钟。 5)当DNA与脂质体进行孵育时，移去细胞原有的培养基并用2ml无血清培养基洗一次，移去用来清洗的无血清培养基。 6)在每一个含有DNA与脂质体混合物的管子中加入0.8ml无血清培养基，轻柔混合，分别把DNA与脂质体混合物加入含有细胞的孔中。 7)细胞在27℃孵育5小时。 8)从细胞中移去DNA与脂质体混合物加入2ml完全生长培养基。 9)将细胞在27℃进行孵育，实验人员必须每日观察，记录转染细胞的生长状态，细胞在转染后48小时长势应该比较良好，但72小时后直至可以看到明显的病毒感染的细胞病变。 2、第一代代病毒的收集与保存 1)当转染的细胞呈现出感染后期的形态时，收集每孔含有病毒的上清，转移到灭菌的15ml 压盖管中。 2)以500*g离心5分钟，从而移去上清中所含有的细胞及大的碎片。 3)将离心后的上清移到新的15ml压盖管中，用来保存第一代病毒，存放于4℃避光保存。 3、病毒的保存 1)病毒存放于4℃避光保存。 2)如果用的是无血清培养基，则加入终浓度为2%的FBS 。血清蛋白可以作为蛋白酶的底物。 3)长期保存时将一部分病毒储存在-80℃用于病毒的重新扩增。 4)病毒的常规储存不要低于4 ℃.病毒经过反复冻融会使其滴度下降10 到100倍。 4、杆状病毒的扩增 1)准备sf9细胞，2*106/孔，在室温孵育1小时。 2)在孵育1小时以后，用倒置显微镜观察昆虫细胞的贴壁情况。 3)在每孔中加入适量的P1代病毒。 4)在27℃进行孵育48小时。 5)在感染后48小时，收集每孔含有病毒的上清，转移到灭菌的15ml压盖管中。以1000*g 离心5分钟，从而移去上清中所含有的细胞及大的碎片。 5、病毒空斑分析 1)准备工作如下： 澄清的杆状病毒保存于4℃ 培养在适当培养基中的sf9细胞（30ml 5*105/ml对数生长期的细胞用于每一个滴度的杆状
细胞转染的操作步骤 转染，是将外源性基因导入细胞内的一种专门技术。随着基因与蛋白功能研究的深入，转染目前已成为实验室工作中经常涉及的基本方法。转染大致可分为物理介导、化学介导和生物介导三类途径。电穿孔法、显微注射和基因枪属于通过物理方法将基因导入细胞的范例；化学介导方法很多，如经典的磷酸钙共沉淀法、脂质体转染方法、和多种阳离子物质介导的技术；生物介导方法，有较为原始的原生质体转染，和现在比较多见的各种病毒介导的转染技术。红外碳硫仪理想细胞转染方法，应该具有转染效率高、细胞毒性小等优点。病毒介导的转染技术，是目前转染效率最高的方法，同时具有细胞毒性很低的优势。但是，病毒转染方法的准备程序复杂，常常对细胞类型有很强的选择性，在一般实验室中很难普及。其它物理和化学介导的转染方法，则各有其特点。 >需要指出的一点，无论采用哪种转染技术，要获得最优的转染结果，可能都需要对转染条件进行优化。影响转染效率的因素很多，从细胞类型、细胞培养条件和细胞生长状态，到转染方法的操作细节，都需要考虑。 一、细胞传代 1. 试验准备：200ul/1mlTip头各一盒（以上物品均需高压灭菌），酒精棉球，废液缸，试管架，微量移液器，记号笔，培养皿，离心管。 2. 弃掉培养皿中的培养基，用1ml的PBS溶液洗涤两次。 3. 用Tip头加入1ml Trypsin液，消化1分钟。用手轻拍培养瓶壁，观察到细胞完全从壁上脱落下来为止。 4. 加入1ml的含血清培养基终止反应。 5. 用Tip头多次吹吸，使细胞完全分散开。 6. 将培养液装入离心管中，1000rpm离心5min。 7. 用培养液重悬细胞，细胞计数后选择0.8X106个细胞加入一个35mm培养皿。8. 将合适体积完全培养液加入离心管中，混匀细胞后轻轻加入培养皿中，使其均匀分布。 9. 将培养皿转入培养箱中培养，第二天转染。 二、细胞转染 1. 转染试剂的准备 ①将400ul去核酸酶水加入管中，震荡10秒钟，溶解脂状物。 ②震荡后将试剂放在－20摄氏度保存，使用前还需震荡。 2. 选择合适的混合比例（1：1－1：2/脂质体体积：DNA质量）来转染细胞。在一个转染管中加入合适体积的无血清培养基。加入合适质量的MyoD或者EGFP的DNA，震荡后在加入合适体积的转染试剂，再次震荡。 3. 将混合液在室温放置10―15分钟。 4. 吸去培养板中的培养基，用PBS或者无血清培养基清洗一次。 5. 加入混合液，将细胞放回培养箱中培养一个小时。 6. 到时后，红外碳硫仪根据细胞种类决定是否移除混合液，之后加入完全培养基继续培养24－48小时。三、第二次细胞传代1. 在转染后24小时，观察实验结果并记录绿色荧光蛋白表达情况。 2. 再次进行细胞传代，按照免疫染色合适的密度0.8X10 个细胞/35mm培养皿将细胞重新转入培养皿中。 3. 在正常条件下培养24小时后按照染色要求条件固定。
RNAi or siRNA Transfection 以24孔板为例，其余规格的转染见表1 1 中板，细胞密度为30-50%适宜。 注意：根据转染后细胞检测时间长短决定细胞中板密度，如果转染后需要长时间后检测，则细胞中板密度适当降低，已避免细胞过度生长导致存活降低。 2 第二天（24-36小时后）每个孔转染方式如下： A 将20pmol siRNA溶于50ul Opti-mem无血清培养基中。 B 将1ul lipo2000溶于50ul Opti-mem无血清培养基中，混匀室温放置5min。 C 将A B两管混合，放置20min。 3 转染期间，将24孔板培养基换成无血清培养基，每孔400ul。将C管mix加入24孔板对应孔中，4-6小时候换成有血清培养基。 Plasmid DNA Transfection DNA(ug):lipo 2000(ul)=1:2-3 转染时细胞密度越高，转染效率，表达效率也越高，并且可以降低细胞毒性。 1 中板。 贴壁细胞：0.5-2X105 cells/well，第二天待细胞密度达到90%以上时转染 悬浮细胞：4-8X105 cells/well，中板后随即转染。 2 转染。 A 将0.8ug DNA溶于50ul Opti-mem无血清培养基中。 B 将2ul lipo2000溶于50ul Opti-mem无血清培养基中，混匀室温放置5min。 C 将A B两管混合，放置20min。 转染期间，将24孔板培养基换成无血清培养基，每孔400ul。将C管mix
加入24孔板对应孔中，4-6小时候换成有血清培养基。 Table 1. Culture Shared reagents DNA transfection RNAi transfection 中板密度*Culture vessel Surf. area per well Vol. of plating medium Vol. of dilution medium DNA Lipofectamine ?2000 cell/well 96-well0.3cm2100ul2X25ul0.2ug0.5ul 0.5-2X105 cell/well 24-well2cm2500ul2X50ul0.8ug 2.0ul 1-3X105 cell/well 12-well4cm21ml2X100ul 1.6ug 4.0ul 2-3X105 cell/well 6-well (35mm) 10cm22ml2X250ul 4.0ug**10ul 8-10X105 cell/dish 60mm20cm24ml2X0.5ml8.0ug***20ul 2-3X106 cell/dish 10cm60cm215ml2X1.5ml24ug60ul *：中板密度根据不同细胞不同实验有所不同，这里仅提的数据仅供参
4. 哺乳动物细胞siRNA转染 4.1 转染方法： 将制备好的siRNA、siRNA表达载体或表达框架转导至真核细胞中的方法主要有以下几种：磷酸钙共沉淀；电穿孔法；DEAE-葡聚糖和polybrene；机械法；阳离子脂质体试剂。目前用的最多的是阳离子脂质体法。 4.1.1 磷酸钙共沉淀 将氯化钙，RNA(或DNA)和磷酸缓冲液混合，沉淀形成包含DNA且极小的不溶的磷酸钙颗粒。磷酸钙-DNA复合物粘附到细胞膜并通过胞饮进入目的细胞的细胞质。沉淀物的大小和质量对于磷酸钙转染的成功至关重要。在实验中使用的每种试剂都必须小心校准，保证质量，因为甚至偏离最优条件十分之一个pH都会导致磷酸钙转染的失败。 4.1.2 电穿孔法 电穿孔通过将细胞暴露在短暂的高场强电脉冲中转导分子。将细胞悬浮液置于电场中会诱导沿细胞膜的电压差异，据认为这种电压差异会导致细胞膜暂时穿孔。电脉冲和场强的优化对于成功的转染非常重要，因为过高的场强和过长的电脉冲时间会不可逆地伤害细胞膜而裂解细胞。一般，成功的电穿孔过程都伴随高水平（50%或更高）的毒性。 4.1.3 DEAE-葡聚糖和polybrene 带正电的DEAE-葡聚糖或polybrene多聚体复合物和带负电的DNA分子使得DNA 可以结合在细胞表面。通过使用DMSO或甘油获得的渗透休克将DNA复合体导入。两种试剂都已成功用于转染。DEAE-葡聚糖仅限于瞬时转染。 4.1.4 机械法 转染技术也包括使用机械的方法，比如显微注射和基因枪（biolistic particle）。显微注射使用一根细针头将DNA，RNA或蛋白直接转入细胞质或细胞核。基因枪使用高压microprojectile将大分子导入细胞。 4.1.5 阳离子脂质体试剂 在优化条件下将阳离子脂质体试剂加入水中时，其可以形成微小的（平均大小约100-400nm）单层脂质体。这些脂质体带正电，可以靠静电作用结合到DNA的磷酸骨架上以及带负电的细胞膜表面。因此使用阳离子脂质体转染的原理与以前利用中性脂质体转染的原理不同。使用阳离子脂质体试剂，DNA并没有预先包埋在脂质体中，而是带负电的DNA自动结合到带正电的脂质体上，形成DNA-阳离子脂质体复合物。据称，一个约5kb的质粒会结合2-4个脂质体。被俘获的DNA就会被导入培养的细胞。现存对DNA转导原理的证据来源于内吞体和溶酶体。
RFect 小核酸转染试剂 货 号：11011: 0.5ml 11012: 1.0ml 储存条件：-20℃ 11013: 1.5ml 产品特点 应 用 产品介绍 RFect 小核酸转染试剂是国际知名科学家崔坤元博士领导我公司研发团队在美国西雅图实验室研发成功的一种新型的小核酸转染试剂。RFect 可用来转染siRNA 、antisense RNA 、microRNA 等200bp 以内的小分子RNA 和DNA ，转染细胞包括绝大多数贴壁生长的细胞，如一般细胞株、肿瘤细胞株等。目前，无论国外还是国内，转染试剂的主要成分均为脂质体或聚乙烯亚胺(Polyethylenimine, PEI)，这两种成分都具有很大的细胞毒性，并且转染效果不好。RFect 采用新型的动物源性的纳米材料，毒性很低并且拥有非常卓越的转染性能。与其它品牌的小核酸转染试剂相比，RFect 的细胞转染阳性率一般在90%以上，Lamin A/C 基因抑制效率在95%以上，而其它品牌转染试剂的细胞转染阳性率一般不超过70%，Lamin A/C 基因抑制效率不超过75%。RFect 细胞毒性很低，转染细胞死亡率不到10%，而其它品牌试剂的转染细胞死亡率一般在30%以上。血清对转染效果没有影响，不必刻意添加或更换培养液。有关RFect 小核酸转染试剂的材料合成和试剂配制我们已申请了国际专利，并通过PCT 覆盖国际上多个国家和地区。 操作步骤：本说明书适用于24孔培养板的转染实验，其他规格的培养板的用量参照下面表格，表格给出的是每孔的用量与体积。 A. 细胞接种：转染前一天接种细胞，每孔 500 μl 培养基（不可加抗生素），使细胞在转染时密度在30-50% 。 B. siRNA-RFect 混合物准备： 1. 6pmol siRNA 用50μl 无血清培养基稀释。 2. 2μl RFect 用50μl 无血清培养基稀释。轻轻混匀，室温孵育5min 。注意：确保在25 min 内执行第三步操作，不要过于延迟。 3. 孵育5min 后，将siRNA 稀释液与RFect 稀释液混合（总体积100μl ）。轻轻混匀，室温孵育20 min 。 C. 将混合物加到培养的细胞内（完全培养基培养）： 1. 将100μl 混合物加入培养孔内，培养孔内含有0.5ml 培养的细胞。轻轻晃动培养板，混匀。 2. 37°C 培养18-72h ，检测基因抑制效果。如果需要，细胞培养4-6h 时可以更换培养基，但不是必须。孵育时间的长短，取决于细胞类型、 所干扰基因本身及分析方法。可设置不同的孵育时间进行实验以确定最佳孵育时间。 转染实验要点： ● 转染过程不可添加抗生素，否则会导致细胞死亡； ● 首次实验siRNA 的用量可稍大（一般10 nM ） ，后续实验根据实验结果修改。 RFect Transfection Reagent Formats for Various Cell Culture Vessels Culture vessel Surface area/well (cm 2) Vol. of growth medium (μl) Vol. of dilution medium (μl) siRNA Amount (pmol) RFect (μl) 96-well 0.3 100 2 x 10 1.2 0.4 48-well 0.8 250 2 x 25 3 1 24-well 2 500 2 x 50 6 2 12-well 4 1000 2 x 100 12 4 ◎卓越的细胞转染性能：细胞转染阳性率高达90%以上，基因敲除效果明显，A549细胞Lamin A/C 基因敲除效率在95%以上 ◎极低的细胞毒性：转染细胞死亡率不到10%，大大降低了因细胞毒性对实验结果的影响 ◎转染细胞范围广，绝大多数贴壁细胞株都能获得比较理想的转染结果 ◎siRNA 转染 ◎antisense RNA 转染 ◎200bp 内的小分子DNA 转染
【试剂与仪器】 1 ．小牛血清。 2 ．双抗溶液（链霉素100μg+ 青霉素100 单位）。 3 ．DMEM 培养基。 4 ．转染试剂（LIPOFECTAMINE 2000 ）。 5 ．细胞培养基（DMEM+10%NCS ）。 6 ．PBS 。 7 ．无血清培养基。 8 ．胰酶（Trypsin ）。 9 ．培养皿，移液管，量筒，恒温水浴箱，离心机，15ml 离心管，微量移液器，荧光显微镜和CCD 。 【操作步骤】 1 ．转染前一天，胰酶消化细胞并计数，细胞铺板，使其在转染日密度为90% 。细胞铺板在0.5ml 含血清，不含抗生素的正常生长的培养基中。 2. 对于每孔细胞，使用50μl 无血清DMEM 培养基稀释0.8μg-1.0μg DNA 。多孔操作可以批量制备。 3. 对于每孔细胞，使用50μl DMEM 培养基稀释1μl-3μl LIPOFECTAMINE 2000 试剂。LIPOFECTAMINE 2000 稀释后，在5 分钟内同稀释的DNA 混合。保温时间过长会降低活性。 4. 混合稀释的DNA （由第2 步）和稀释的LIPOFECTAMINE 2000 （由第3 步）。在室温保温20 分钟。注意：溶液可能会混浊，但不会影响转染。复合物可以在室温保持6 小时稳定。 5. 直接将复合物加入到每孔中，摇动培养板，轻轻混匀。注意：如果在无血清条件下转染，使用含血清的正常生长培养基进行细胞铺板。在加入复合物前移去生长培养基，替换为0.5ml 无血清培养基。 6. 在37℃，5 ％的CO 2 中保温24-48 小时，无须去掉复合物或更换培养基或者在4-5 小时后更换生长培养基也不会降低转染活性。 7. 在细胞中加入复合物24-72 小时后，分析细胞抽提物或进行原位细胞染色，检测报告基因活性。这依赖于细胞类型和启动子活性。对稳定表达，在开始转染一天后将细胞传代至新鲜培养基中，两天后加入筛选抗生素。进行稳定表达需要数天或数周。