Aplikovaná chemie a biochemie Přednáška č. 4 Manipulace genové exprese Modulace exprese nebo funkce proteinů: (3) (2) Small-molecule Overexpression inhibitor system _|_ (4) mAb Protein »- RNA degradation (e.g. RNase H) Modulation of splicing Arrest of translation Table 1. A comparison of different experimental approaches for modulating the function of cell signaling molecule Antisense oligonucleotide Fig. 1. Experimental approaches that are curre of a particular signaling molecule to determine knockout; (2) overexpression systems; (3) small and (5) antisense technology. The advantages a text. Antisense technology uses chemically mod target RNA sequences through a Watson-Cric of adenine-thymine (A-T) and guanosine—cytc Method Versatility Specificity Required resources Cost Probability of success Potential for drug developme Overexpression Low to moderate Moderate Moderate Low Moderate Low systems Gene knockouts High High High High Moderate None (mammalian) Small-molecule Low Low High High Low Yes inhibitors Monoclonal Low High Moderate Moderate Moderate Yes antibodies Antisense High High Low to moderate Low to moderate High Yes oliaonucleotides • chemická inhibice, aktivace • změna genové exprese • selekce rezistentních klonů • použití přirozených mutantů • overexprese proteinu • overexprese dominantně negativních mutantů tranzientní vs. stabilní transfekce • použití antisense oligonukleotidů • RNA interference - tranzientní a stabilní Chemická inhibice, aktivace: Forskolin inactive PKA regulatory inactive subunit catalytic subunit complex of cyclic AMP and regulatory subunits active catalytic subunits Figure 15-32. Molecular Biology of the Cell, 4th Edition. CAMP PACAP Fig. 1. Activation of an estrogen-responsive model promoter in aT3 cells by cAMP. Cells were transfected with 2 jig ERETkluc reporter plasmid as described and treated with vehicle (Con), 10 iiM 170-estradiol (E2), 1 pM FSKh 2 mil 8-Br-cAMP (cAMP), 200 n.\[ PACAP, or 100 ng/ml EGF for 24 h. Data shown represent the normalized mean ± sem from five to seven independent experiments performed in duplicate. Significant differences from control are denoted with asterisks (*, P < 0.05; »*, P < 0.01). Á. * 0 ■ Vehicle DFSK-7M QFSK-6M 1 ťj irů (j lxJO" 5xKT B. Dtjst H«9 (M) Různá specifita inhibitorů: 11 Inhibibon of protein kinases by commercially available inhibilors hiülor concentrations used ane shown in paremheses. Resuhs are presented as kinase activity as a pencenlage of thai in control Incubations (averages of duplicate determinations). ATP was present al 0.1 mM in all assays. Activity!% of control) nag V 27632 H Al 077 Roltlerin KN&2 U 0126 PD 164352 PD 96059 SB 203530 SB 202190 Wortmannin IY 294002 Quercstin Rapamycin LiCI KCl (10 ^M) (10 /M) (20>M) (20 /i M) flO/íM) (10 fill) [io pW (50 {i M) (10/M) (10 pM) (1 /M (50 pM) (20 f M) 1.1 /.Mi (10 DM] (10 r, 90±4 103 + 0 89 + 3 106 + 2 101 ±1 56+1 5+1 69 + 1 99+1 93±1 96+1 101+1 94+3 99 + 1 116±1 109; 87 + 1 94 + 3 94 + 8 139 + 17 92 + 1 92+3 107+5 65+3 85+6 39 + 6 90+12 114 + 13 113 + 8 90 + 16 107+2 102; 97 + 1 96 + 1 96 + 1 49+1 104+4 96+4 102+2 111+2 101+5 93+1 97+1 106+0 101+5 98 + 6 92 + 0 B9- 99 + 1 94 + 5 93+3 111 + 5 95+6 75+1 100+3 65+5 2+1 O+O 36+5 96+3 138+7 93 + 12 108 ±1 102- 97±4 107 + 3 97±15 127+3 98 + 1 90±4 119+1 95+6 10±1 3±0 74+3 96±0 150+6 88 + 10 96 + 1 104; 106 + 2 1O0 + 3 87+2 146+4 95+1 100+2 100+1 96+2 96+2 ŮO+2 75+5 97+2 132+1 100±1 99 + 1 108; 105 + 3 95 + 1 1113+11 130 + 1 110+22 111+1 98 + 3 94+8 93+4 37±9 79+11 94+1 103+3 82±12 84 ±2 99: 16+1 72 + 13 37±6 79 + 1 69 + 16 88±3 66+3 93 + 9 83+10 95±8 92±3 70+2 20±3 95±17 95±2 7B: 99 + 12 99 + 3 90+1 5 + 2 59+5 102+2 96+6 95+6 93+2 97 + 1 102+5 74+5 90+3 125 + 7 72+4 9B; d±D 57 + 2 19 + 2 33 + 3 61+10 104+1 116+3 66+3 86+2 88±3 99±4 83±7 37±3 104 + 2 104±6 105- 81+4 104+11 91 +19 6+5 36 + 1 93+3 71 ±4 106 + 6 112+4 83 + 11 35+4 66+6 51+2 74 ±10 76 + 3 104; CiP TŠTT 91+2 35+2 17+3 94+9 95±2 105+1 106 + 4 96 + 4 66±9 97±5 91+4 104+6 1O4 + 0 96+1 96; 96 + 0 66+1 95 + 2 95+5 92+2 99+1 93 + 4 89+4 92+0 100+2 91 ±4 70+1 99 + 5 98 + 1 97; 104+3 115+12 92+2 36+2 70+7 99+1 65+2 66 + 4 89+4 67±9 3fl±9 76+4 81 ±4 110 + 5 105±1 9B; 17 + 1 90 + O ae+5 27+3 67 +a 79+5 69+4 62 + 1 62+1 53+2 96+6 60+2 99 + 2 91+0 95 + 5 96- 25 + 1 109±5 92 + 5 81 + 0 76+5 91+1 111±14 90 + 1 83+5 9Ů + 1 101 ±7 72+1 35±0 108 + 6 99+1 100- 94 + 3 32 + 1 93+7 93 + 5 92±3 66+0 100+2 87+1 75±1 106±3 31 ±7 25±0 109 + 2 95±2 101: 107 + 2 92 + 9 90+5 13 + 1 38 + 4 105 + 3 B3 + 3 101 ±1 66+3 61+6 35+10 53+1 30+1 89 + 9 58+3 99: CHID 15±T 13 + 2 7+1 63+7 66+0 94+1 107±4 60+3 77+1 61 ±2 91 ±1 104±10 55±2 92±2 101+2 102; 95 + 0 77+1 93 + 1 97+0 85+4 69 + 3 97 + 4 96 + 2 94 + 0 106+1 103+0 16+0 106 + 0 106+1 105- 104±2 96 + 3 102±3 103 + 6 103+O 107+1 96+1 67 + 3 97+1 93+1 90 + 3 16+1 19+3 104±7 73 ±5 112: 51+3 81 +9 56+1 63+3 106+3 101+1 117+15 67 + 4 104 + 13 91 ±1 100+4 44±10 32+4 103 + 4 96 + 2 93: 76 + 6 109 + 0 94±0 70+3 92 + 1 87±1 99+5 65+8 32+3 37±0 95±4 35±8 63+11 102 + 2 99±4 105; 21 ±1 99 + 1 82 + 6 93+1 93+1 107+3 101+1 104+2 O+O 96+3 95 + 1 104+3 99 + 4 95+2 95+1 99 + 3 104 + 1 4+1 90±2 56+1 102±2 96±3 97: 95±1 114+4 94+1 109- 80±3 81±3 87±2 94- 86 + 1 79+1 108 + 5 113: 91±2 89±1 100+1 102; 0+0 13+0 18+2 6 + 1 15+1 Ďalší kritické body pro použití inhibitorů: • rozpustnost; • stabilita; • biodostupnost; • nežádoucí reakce s receptory; • různá aktivita vůči izolovaným nebo rekombinantním proteinům a v buněčné kultuře nebo v in vivo podmínkách. • selekce rezistentních klonů • použití přirozených mutantů nebo linií KO myší; Buňky jsou dlouhodobě pěstovány v přítomnosti účinných koncentrací vysoce toxických látek - např. cytostatik - jsou vyselektovaný přežívající buňky schopné růst v přítomnosti toxických látek - např. cytostatika, toxíny. Zpětně jsou pak studovány změny na úrovni exprese proteinů. Percent of Control O X O o< o < Sľ a) a) 3 a>> Q. O O < Využití buněk izolovaných z knock-out myší: MEFs (mouse embryonic fibroblasts) C) p3S+'+ p38-'- BPDE-2 (\M ) a .25 .5 i o .25 .5 1 Cytosolic Cytc *«.^»^.- . _ _ PARP-1 Manipulace s funkcí proteinu prostřednictvím overexprese identického genu se změněnou funkcí: Loss -of-f unction mutations ■ Null mutations - completely lacks function of gene ■ Infer function of wild-type gene Hypomorphic mutations ■ Partial loss-of-function ■ Infer function of genes expressed during different times in development Conditional mutations ■ Cause loss-of-function only under special circumstances ■ E.g., temperature sensitive mutations ■ Infer function of gene at different developmental stages Dominant-negative mutations ■ Mutant allele counteracts wild type allele in heterozygote ■ Also haploinsuffíciency - mutant allele is dominant in heterozygote because two wild-type alleles required for development podmínkách in vitro můžeme vhodný model získat buď z existujícího rqanismu, nebo připravit gen kódující změněnou funkci uměle. bHLH UŕCAE (325-329) PAS Í Q-rich AHR B&&Š illiiiiliil KOM 1 121 374 805 A323^94 Ä323-607 SSr Fvy^rrr «liiiif.inii.iiinii EM A495-805 5öööl:iV:.!:l.ls!; Transformace kompetentních bakterií (E. col i) - tepelný šok - elektroporace Izolace jri^i|>^-<š><----ä^B Comments f ar pcDNAl: 4033 base pairs Col E1 origjn: bases 1-537 M13 origin: bases 588-1182 SupF gene: bases 1183-1384 CM V promoter: bases 1517-2170 T7 promoter: bases 2171-2189 T7 primer sequence: bases 2170-2169 PoMinker: bases 2187-2306 5p6 primer sequence: bases 2307-2325 S pli c e and poly A: bases 23 26- 3 024 PoJyims. crign: bases 3029-3870 5 V4d or gin: bases 3371-4Ü33 plazmidu Transfekce buněčných linií in vitro: Transfection is the process of introducing naked DNA molecules into cells. Transfection can be categorized into 2 major types, stable and transient. Transient transfection is temporary and high level expression of foreign genes. Expression lasts for several days, but is lost as the DNA newer integrates stably into the host cell DNA. In contrast, stable transfection occurs with a lower frequency (10 to 100-fold lower), but expression is maintained for the long term because the foreign DNA does integrate into the host DNA.In the case of stable transfection, cotransfection is often used to introduce a selectable marker (such as an antibiotic resistance gene). Since only one in 1000 cells might be stably transfected, it is necessary to select these cells fromthe total population. Cells that express the selectable marker also take up and express the other gene of interest. Lipofection is a procedure in which the DNA is complexed within lipid droplets. The droplets interact directly with the cell membrane and fuse. The DNA is liberated into the cytoplasm and some eventually reaches the nucleus. Lipofection is one of the most efficient methods of transfection. However, it is also relatively expensive, it can be toxic to cells, and it cannot be used with cells growing in serum. Lipofection, like other forms of transfection, works much more efficiently if the cells are rapidly growing. This is because the nuclear membrane is absent during cell division, allowing easier access to the host DNA. Calcium phosphate is another popular method for transfection. In this procedure, the DNA is precipitated with calcium phosphate aggregates. The cells phagocytize the aggregates and the DNA is released into the cytoplasm and eventually reaches the nucleus. This is the oldest method and its main advantage is that it is cheap and easy to perform. However, calcium phosphate transfection is not as efficient as lipofection and the precipitates often cause cytotoxicity. Electroporation is another method of transfection in which cells are exposed to an electric shock. This induces transient aqueous channels in the membrane for DNA to enter the cytoplasm. On the positive side, electroporation is rapid and simple, and it works on almost all types of cells. However, one needs special equipment such as an electroporator to shock the cells. The transfected cells also have high cytotoxicity after shocking. There are several viral vector systems that have been developed for the study of gene expression in vitro or in vivo, including recombinant vaccinia viruses, retroviruses, and adenoviruses. Due to bio-safety regulations, a special lab facility must be available. Metody selekce Prokaryota Antibiotic Formula Ampicillin C^l ligNjÜ4 SNa Ampicillin is a semi-synthetic penicillin derived from the penicillin nucleus, 6-amino-penicilknic acid. It causes cell death by inhibiting cell wall biosynthesis. Resistance to ampicillin is mediated by ß~lactamase cleavage of the ß-lactam ring [bia gene). Klinomycin Sul lote Kanamycin is effective as a bacteriocidal agent by inhibiting ribosomal translocation and eliciting miscoding. Resistance is confened by the KanR-TnS gene product (aminoglycoside phosphotransferase), which modifies the antibiotic and prevents interaction with ribosomes. Liquid kanamycin (1G0X) contains 10 mg/ml kanamycin (base) utilizing kanamycin sulfate in O.S5% saline. C18H3ůN4Ou-HaS04 Tetracycline Tetracycline is a bacteriocidal agent that inhibits protein synthesis by preventing binding of aminoacyl-tRNA to ribosomes. Resistance is conferred by the Tet^TnJO gene product (an inner membrane protein that effluxes the antibiotic), which blocks cell wall permeability. ^H24N2Oa •iici Eukaryota Geneticin® He La 200-400 * pcDNA3.1™ vectors (Constitutive mammalian expression) NIH3T3 600-1,000 * pIND vectors (Ecdvsone-Inducible mammalian expression) CHO - 400 * pShooter vectors (Intracellular protein targeting) 293 HEK 600-800 * pDisplay™ vectors (Protein display) Jurkat T cell 600-700 * pVP22 vectors (Protein translocation) * pBlue-TOPO* pGlow-TOPOu vectors (Assessing promoter activity) Hygromycin B He La -550 ♦ pcDNA5 vectors (Constitutive mammalian expression) CHO -250 • pIND/Hygro vector (Ecdvsone-Inducible mammalian expression) Blasticidin S He La 1-3 ♦ pcDNA6 vectors (Constitutive mammalian expression) NIH3T3 5-10 • BsdCassette™ vectors (Constructing customized Blasticidin- CHO 5-10 resistant vectors) COS-1 3-10 • pIB/VS-His-TOPO® vectors (Stable insect expression) 293 HEK 5-10 * pMIB/V5-His vectors (Secreted insect expression) S2 Drosophila ~S ♦ pCoBlast (Selection vector for DES®) S. cereuisiae -25 Overexpression/ectopic expression: • exprese velmi vysokých hladin proteinu v buňce, která ho za normálních okolností neexprimuje, nebo jen v omezené množství; • nevýhodou je častá nespec if ita - aberantni lokalizace proteinu, aberantni interakce s dalšími proteiny, atd. Inducibilní exprese: O = 00 -_- > _ i— -n ť-- — ív1^- ní ^ —----cj— n, Cdkl-cyclin B Cdkl cyclin A Cdltí-cyclin A CdU-üynlin D Cdke-cyclin D C[Jt2-nynliri E Comments for pcDNA'"4/T0 507E nucleotides CM*/promoter: bases232-95fi TATA box: bases BOAS 10 Tetracycline operator (2X TetQ;) sequences: bases 320-359 CMV forward priming site: bases 769-709 Mulli pie cbn ing site: bases 967-1077 BGH reverse priming site: bases 1069-1106 BGH poryadanylation sequence: bases 1095-1319 fl origin: bases 1365-1793 SV40 promoter and origin: bases 1303-2143 EM-7 promoter: bases 2133-2249 Zeocin"" resistance gene: bases 2250-2624 SV40 early poiyadenylation sequence: bases 2"54-2BB4 pUC origin: bases 3267-3937 Wa promoter: bases 4937-5041 (complemerilaiy strand) Ampicillin (We) resistance gene: bases 4062-4942 (complementary strand) Time (h) Doxycyclin 0 24 48 + + DNA synthesis (induction x-fold) D Ö Ö CD Ö T l Cyclin A-> _ — «M ■ ERK2+ 9. ■ + Doxycyclin (48 h) + TCDD(48h) Dominantně-negativní mutant - definice: - mutantní protein, který potlačuje funkci wt proteinu v případě společné exprese; - mechanismy - mul timer izace, titrace (upstream or downstream targets), aktivní represe. signal mul »cul« nemal protein mutant complex of mui ani in complex protein prolein and normal ;:í i ľr'i l'i - > protein ACTIVE INACTIVE INACTIVE signal mulucule •i ■• i il tyrosine U IHM domain inactive receptor tyrosine kinases kinase sei I vlly stimulated bv eross-phospticif v'»l ion O o nokkw activation Konstitutivně-aktivní mutant Full length AhR bHLH V PA5 domain / .....*l I CA AhR-GFP C iK^H CHrich 1 1 EGFP B CVP1A1 GAPDH ď Antisense (2) Splicing arrest Antisense oligo Intron trends in Pharmacological Sciences Fig. 1. Experimentally demonstrated mechanisms by which antisense oligonucleotides disrupt protein synthesis include: (1) steric blockade of ribosomal subunit attachment to mRNAat the 5' cap site; (2) interference with proper mRNA splicing through antisense binding to splice donor or splice acceptor sites; and (3) RNase-H-mediated degradation of hybridized mRNA. The latter can occur anywhere in the mRNA where an antisense molecule binds with sufficient affinity, including the 5' and 3' LJTRs, at the translation initiation codon, and in exons or introns. Table 3. Antisense oligonucleotides currently in clinical trials or on the market Compound Protein target Indication Sponsoring Development company phase Vitravene CMV IE2 CMV retinitis Isis/Ciba vision Approve (ISIS2292)3 ISIS2302 ICAM-1 Crohn's disease, organ transplant, psoriasis Isis Phase II ISIS3521 Protein kinase C a Cancer Isis Phasell ISIS5132 RAF kinase Cancer Isis Phasell G3139 RCL2 Cancer Genta Phasell INX3280 MYC Restenosis INEX Phase II G EM 132 CMVUL36 CMV retinitis Hybriden Phase ISIS2503 Ha-RAS Cancer Isis Phase II IS IS 13312 CMVIE2 CMV retinitis Isis Phase GEM92 HIV AIDS Hybriden Phase GEM230 Protein kinase A Cancer Hybridon Phase aVitravene (Fomivirsen, ISIS2922I has been approved for the second-line treatment of cytomegalovirus (CMV) retinitis in patients with AIDS who are intolerant uf or unresponsive to previuus treatment(s) for the disease". All drug compounds are phosphorothioate oligodeoxynucleotides except ISIS13312|2'-methoxyethyl), GEM92 [2'-methoxy| and GEM230 (2'-methoxy), which contain 'second-generation' 2'-sugar modifications. Abbreviations: ICAM-1, intercellular adhesion molecule 1; IE2, immediate early gene 2. UPS- April 2000 (Vol. 21) 1 45 505� uesign a príprava anrisense ■H-o of x o=p^ÔJ o BASE (Y) BASE \ Phosphodiester ■vv-o o BASE (Y) BASE Phosphorothioate (a) 2'Sugar modifications (X) Fluoro Met noxy —o Propoxy ^ CH.3 Methoxyethyl (b) Heterocycle modifications (Y) 5-Methy! cytosine ^Cy y* 5-Propyne cytosine # ■»N }—NH; h"' Phenoxazine h" Tricyclic-cytosine (G-ciamp) —H )- -Q y -NH2 (c) Backbone modifications (Z) Methylene- W^-°~V*B methylimino A___f N - I I Base H3C' N CH3 O-P-N Morphlino o^ cHj ^r" 3" K Methylene i—l H3C R |išio| W^yBase Base y \ Peptide nucleic acid (PNA) K" '—NH y^ o 3' 0 /H ířmiís íit Ptinrmíttxlogicat Sciences Fig. 3- Structures of chemical modifications that áre employed in antisense technology. Phospliodiester oligodeoxynucleotides (Jplam DľiA'fare not useful in antisense technology because of rJieir inherent susceptibility to nuclease degradation. Chemical modifications involving substitution within the {a} sugar, (b) heterocyde Ibasel Or (c| backbone subslituents of DNA were designed primarily to reduce nuclease sensitivity, improve affinity for RNA hybridiža t lůn. Or both. Some of these modifications also provide pharmacokinetic and lOitrcülogical *advaiil-ages. PrKísphurutliiti-aHryliy-u-JĽUÄyMucItĽUklĽĽ urt; s'jmeriinťs leícrriÄl tu uíí 'first-generation' antisense oligonucleotides whereas oligonucleotides containing other modifications are referred to as 'second-generation. (See references for a description of the chemical and biological properties of the modifications indicated"-*4 A) ongonuKieoTiau. 1) Chemistry - unmodified phosphodiester DNA is rapidly metabolized inboth serum and cells - several chemical modifications can be incorporated into antisense molecules to boost their nuclease resistance. Two examples are phosphorothioate oligodeoxynucleotides and 29-<3-methyl oligonucleotides. 2) Length - most antisense molecules are 15-20 bases long, a length theoretically sufficient to pick out a unique sequence from others in the human genome and identify a target mRNA (Ref. 18). Antisense oligomers of this size have been successfully used to discriminate between two gene products that differ by a mutation of a single bas. Longer oligonucleotide sequences (e.g. 30 nucleotides) aremore expensive to synthesize and they might actually increase the risk of non-sequence specific mRNA cleavage because of growing probabilities that other mRNA hybridization sites will be included in long oligomers. Shorter oligomers, meanwhile, generally do not have sufficient affinity to result in adequate potency. 3) Sequence selection - empirical - not all areas of a mRNA molecule are equally amenable to antisense hybridization. The reasons for this are unclear but probably involve mRNA secondary structure, proteins bound to the mRNA or accessibility of hybridized mRNA to RNase H. 'gene-walk' approach involves synthesizing oligonucleotides that target regions scattered throughout the entire mRNA sequence and then evaluating these compounds in cell-culture assays RNA Interference RNA molecules have been used for over two decades to reduce or interfere with expression of targeted genes in a variety of systems. Historically, these methods have been called post transcription gene silencing (PTGS) in plants, quelling in fungi and RNA interference (RNAi) in higher animals. Although originally thought to require use of long double-stranded (ĎS) RNA molecules, the active mediators are now known to be short DS RNAs. These short interfering RNAs (siRNAs) are naturally produced in vivo through nucleolytic processing of long ĎS RNAs. Short DS RNAs can also be chemically synthesized and used to experimentally inhibit gene expression. The Nobel Prize in Physiology or Medicine 2006 for their discovery of RNA interference -gene silencing by double-stranded RNA" Andrew Fire Craig Mello Figuře 5 A model ro r the mechanism of RNAi. Silencing triggers in the form of double- certainly, such complexes act by promoting RNA degradation and translations stranded RNA may be presented in the cell as synthetic RNAs, replicating viruses or may inhibition, However, similar complexes probably also target chromatin remodelling, be tnanscnbed from nuclear genes, These are recognized and processed into small Amplification of the silencing signal in plants may be accomplished by siRNAs priming interfering RNAs by Dicer. The duplex siRNAs are passed to RISC (RNA-induced RNA-directed RNA polymerase (RdRP)-dependent synthesis of new dsRNA. This could silencing complex), and the comp lex becomes activated by unwinding of the duplex, be accomplished by RISC-mediated delivery of an RdRPor by incorporation of the siRN/ Activated RISC complexes can regulate gene expression at many levels, Almost into a distinct, RdR P-containing complex, IMuľtigene family or Foreign DNA (naked) repetitive transgene ™99er jjnmrrffin jjjarnTjTJiiri Transgene CH, "* ■ • ■ r * Transgene assembly/ DNA/chromalin •nodification * L TjJJTjrnffirTI s^sTrna TTTTTTTTTTTTTTT Foreign dsRNA Developmental or expe mental transcription ^ftnmmjiii jjjjqtjQ shRNA üO pre-miRNA Intermediate Silencer -rrrrrr mm siRNA \ 'TTTm~ TTTTTT ...... _LLLli I TTTTTT _^ TTTTT linn ...... IHIII siRNA urn miRNA MIHI rnrrr nnTnT ■ '''■ 111111 "im I A' r\ ciHj Target RITS Rrr Heterochrarrtatin formation and transcriptional silencing -----AAAA mRNA destruction or translational repression Figure 2 Model depicting distinct roles for dsRNA in a network of interacting silencing pathways. In some cases dsRNA functions as the initial stimulus (or trigger), for example when foreign dsRNA is inlroduced experimentally. In other cases dsRNA acts as an intermediate, for example when 'aberrant' mRNAs are copied by cellular RclRP. Transcription can produce dsRNA by readthrough from adjacent transcripts, as may occur for repetitive gene families or high-copy arrays (blue dashed arrows). Alternatively, transcription may be triggered experimentally or developmental^, for example in the expression of short hairpin (shRNA) genes and endogenous hairpin (miRNA) genes. The small RNA products of the Dicer-mediated dsRNA processing reaction guide distinct protein complexes to their targets. These silencing complexes include the RNA-induced silencing complex (RISC), which is implicated in mRNA destruction and translational repression, and the RNA-induced transcriptional silencing complex (RITS), which is implicated in chromatin silencing. Sequence mismatches between a miRNA and its target mRNA lead to translational repression (black solid arrow), whereas near perfect complementarity results in mRNA destruction (black dashed arrow), Feedback cycles perm it an amplification and longterm maintenance of silencing, CH3, modified DNA or chromatin; 7mG, 7-methylguanine; AAAA, poly-adenosine tail; TGA, translation termination codon, Kritické body pro aplikaci siRNA: • vhodně navržená sekvence (w ti; 3 rn); • stabilita siRNA; • vhodný způsob transfekce; • optimální stav buněčné kultury; • především optimálně nastavený systém kontrol a vyloučení tzv. nespecifické interferonove reakce. Praktické využiti syntetické siRNA: AhR control siRNA DsRed siRNA AhR DsRed siRNA siRNA AhR * MM M - • CYP1A1 cyclin A ERK2 3 RNA Interference pomocí shRNA Target Synthetic siRNA against CDH1 ssousncs A Z ^ T<^ 5'-UGAGAAGUCUCGCAGUCAGTT-3J | ^ " - - TÁČÚ ČÚÚéÁGftGGGĽI ČÁGCJ Č- 5' v 7 \a . \ / Predicted transcripts against CDM1 A ^.Q -gff.ŕ— 5'-UGAGAft.GUCUCCCAGUCAGC GA v újr ^S\ ^'-uuÁčyčuúcÄGÄGGGÚcÁ-iučucG CDHl H \\ 5'-UGAGAňGUCTICCCAGUCAGu a í í pSUPER ; s n ni n ,. u j Cyc-D1 \\ // 3' -UUÁČJČUucAgaGGGUcAGtÍČaG & t? m OJ í pSUPER- £ £ ^ CDH1 g 8 a irrra 1 2 3 4 5 6