Molecular Biology I
Molecular Biology I MBioS 503
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This 97 page Class Notes was uploaded by Carol McDermott on Thursday September 17, 2015. The Class Notes belongs to MBioS 503 at Washington State University taught by Raymond Reeves in Fall. Since its upload, it has received 45 views. For similar materials see /class/205937/mbios-503-washington-state-university in Molecular Biosciences at Washington State University.
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Date Created: 09/17/15
Friday Oct 24 310 1A EME B46 Oct 3 1 310 4 p m Fr 2008 Transgernic Cells and Organisms 1 Tran sf e ction Techll iq ue s r0 aches 21an Utility ea J1 xpres 531011 an cl Selection bystems iGene Targeting in Highe r Eu39kary39tes c 39G en e P re r 4 1 c y ally s i s G en e d ilencing Te c39hll iue S ells Why Bother o Gene Knockoutlenockiniseplacements Molecular Genetic Studies OverUnder Expression of Genes Transgenic Animals Gene Therapy Mis expression Mutation Analyses Two Types of Transfections Transient Stable Short term 1296 hr Permanentfgenerates a new cell line No S39e ti quot Requires selection requ39red All cells have Generally a small transfected DNA percentage of cells Production of a stable cell line requires a Dominant Selectable Marker Non LVlutant LVlammallan p ellS Bga cterial gen e coding for neomycin phosphotransferase N ejo confers resistance to aminoglyco sid e antibiotifcs kanamycrin n e39omycin D Which are proteins synthesis inhibitors V Resistancre gromycin Br is an aminocyclit ol that inhibits protein synthesis by disrupting promoting mistra nsl a t ion bqaoteria l gromycin B te ras e gene detoXifie s is used as 39a selectable marker non mutant cells bacterial analogue of mammalian HGPRT In contrast to HGPRT however it h as the additional ability to convert Xanthine to xanthine XMP and hence39 ultimrately to can used to as a quotsele39ct able markerquot Ior non mutant Cells in a 1neac11u atlcniinc IIIyClhenilic lcil and xlntljinc ys Irll39llpcltlogrly OT urinei OIquot Pyrimidine Nucleotide Biosynthetic F39orcaeis Uitili Z ati on of Purine or Pyrimidine Salva39e Pathwa rs for Nucleotid e S nth eslis P ri nl il I nos TCU r 3 39 Purine l 3le jig End ogenous 143 1211 0 enOIIS 7 LG lutamine Glyclne l I inI il ille Salvage 3 gt 11gter 4quot 1 I m I Thymidine I I cytosine Gu an lne P W dPu TP Urirdin e I n o sih e I V I Hyboxanthing L Xa39nthine ba cteri i quot Nu cl Cyt39oplasjljrl Cell Mem b Othet icra l C ell lzective I n hi bition of P u ri he or ri m i d i ne N u cleoti de Bi osynth39eti c Forces Utii lizatiOn of Purine or Pyrimi dvi ne Salvage PathWays for 39Nucrle oti39de Synthesis Asparrate Tetrahydrofolate Dihydrofolate NADP Blocked by aminopterln 77 77777 77 or amethopterin reductase DHFR Dh d OM I t N ADPH Hquot methotrexate l y r o a e Salvage pathway Thym idin e kinase TK Thymidine dTMF dl FP gtDNA 39 h mquot n Kinase Salvage Pathw y and Exogenous Thymidine to Survive The a e nova and salvage pathuvav dTNIF blocked L can dependan on Ehyrnicfine and H11 8 lnopl e i111 n aaking the rhidine kjnas de novo salvage 45 N1oimethylene tetrahydrofolame JIJMP ATP Wthyr nndvne hyihndylate Synthelase retrahydroforate Imhydrofolalzi educzase L uTMP ADP n dlhvdrcfolate thyrr ndlne 1 klnase quotlt Mammalian Cells oHypoxanthine Guanine Phosphoribosyltransferase mutants HGPRT Select for by resistance to toxic guranine analo gs like thioguanine or azaguanine HPRT c ells are l illetl in HAT medium see below oAxienine Mutant gt quotSelec ted for re39s1i39sta7nce to tOXic39 gadeni ne anzalogs 2 flu oroadenine 2 6 diaminopnrine cells l illetl in the Of azaserine L Ki39n ase mutants SEe19 c teid for by resistance to toxic thymidinfe ratnratlpgs Such as BUdR TiK cells are kil lccl in HAT medium contains ypoxanthine Phenotypes of TK and TK thy midine kinase cells in various growth mediLLrns PLAT medium contains hm 39 39 39 d t Hypoxanthine is included in the HAT medium to bypass the block in the de novo path of purine synthesis xvhich also requires tetrahydrofolate TK Cell and L hymidine TK Ce R I 7 Acetic for BUdR Resistance Transformakion of TK mouse cells to TK by chicken DNA A restriction endonuclease digest of toEal chicken DNA is used to cransfect mouse TK cells growling in culture After the transfection period the cells are placed in H xl medium Cells that received no chicken DNA or chicken DNA Fragments that do not contain the chicken thymidine kinase gene fail to grow The rare transformed cells that integrated a chicken DNA Fragment containing the gene into the mouse genor ne grow and forrn colonies Note that if the restriction endonuclease cleaves the Chicken thymidine kinase gene no transformed Cells appear midine Kinase TK Gene b IIAT Selection restriction endonuciease TK gene total chicken genomic DNA mouserTK Ce e in norrnlgi grow th medium change to HAT medium puninn Biosynthetic Pathways Endogenous pathway Glycine Terrahydrofolate Dihydrofolate DHFR IdiNADiPiii reductase I V 39 Dlhydrofowlate Salvage pathway Adenine phosphoribosvltransferase I m1F w AIJ39 AT APRT 39 id Adenine Blocked by Hypoxan tn rte Hyocxanrhine W LJVLQQ guanine phosph orinosyltransferase XGPR E JJ LNot mammalsj Hypoxant e guar une phosnho bosyllransferase HGPFI r ar rh quothe Guanine Biockad bv aminopterin octerin O r a m at h ihio Le9xa re sz13 lr lb1z tj xvuoxdrmthlr e Im lblfjr IjaDScr Ice Gr guanznex hypoxamnnme G nquot4 F3 p pp in guanun X GP lK39I e x phosphoribcslt 1rar1s eres xaquot39ltV I1r r Approaches for Gene Delivery Getting Past the Cell Membrane Biochemical Calcium Phosphate CoI reci itation Liposomes Physical Microinjection Electroporation Biolistic Particle Delivery Infections Retrovirus Adenovirus Adeno associated virus Approaches for Gene Delivery Biochemical Calcium Phosphate Co precipitation Liposomes Charge is the Key Approaches for Gene Delivery Biochemical Calcium Phosphate Coprecipitation Endocytosis 9 q E3 Approaches for Gene Delivery Biochemical Liposomes Membrane Fusion Approaches for Gene Delivery Physical Microinjection Electroporation Biolistic Particle Delivery Disruptions of Membranes is Key Approaches for Gene Delivery Infections Retrovirus Adenovirus Adenoassociated virus Advantages Highly Efficient Disadvantages Requires Packaging Cell Line and Laborious Differences in Viral Delivery of Transgenes Adenovirus ds DNA Episomal ds DNA element Retrovirus 33 RNA Random integration into chromosomes Adenoassociated Virus 33 DNA Potentially selective integration into chromosome 19 human AdvantagesDisadvantages of Viral Vectors o Retrovirus Requires dividing cells exception Lentivirus Random Insertion Limited insert size 8 Kbp Stable expression 0 Adenovirus Works with dividing and nondividing cells No integration in host genome Relatively large insert size 35 Kbp Transient expression 0 Adenoassociated Virus lnfects dividing and nondividing cells Potential for selective insertion currently random Limited insert size 45 Kbp Stable expression Difficult packaging strategies Demonstration of CeII S pecific PromoterEnh ancers IS39olation of First Experiments Demons rating fell Specific PromoterEnhancers Endrocrine B cell Insulin gt ICAT Ho CAT CAT Endrocrine Enzyme B Cel Activity Exocrine Cell Chyrnotrypsin 3 mm a Exocrine CAT Cell Enzyme Activity Ref Walker et al 1983 Cell slecific expression controlled by the 5 flar1kir1g region of insulin and eliyinotrypsin genes Natllre 306 557 61 Initial S e rva tio n s I 0111 39c39uxltukre normal cells as a exvhi bi39t CdnjtaVc t Ilihibition of Growth cells used as a model norm alquot cell system although are in fact l immo rtalize dquot 0111 culture can39ce rous O39r neoplastically transform39ed c elIs gl OW as multilayered 01quot ifOC iquot 0 o s 045 tiger4 39quot N g Human tumor cells growing in vitro J Purify tumcr ltequot DNA very w 1 Shear W 30 50 kb DNA Calcium phosphate precipiicfion 00quot woo ngeo V 9 6 NilU373 mouse cells Trans eltf DNA wait 2 weeks Jr J Repeat procedure Tertiary fransfecfants Etc Isolate human DNA from mou cells fragment contains the ras oncogene I OMost active oncogenic ras genes result form a single base pair change that leads to a single amino acid change Cily12 or Gln61 v Five or more 1 t ras genes 1 humans Point mutations in three of these are to b e causally related to canc erous transformation H ras sarcoma rvi rus derived ras K ras Virus derived Ijas N rasr Neurcb1astoma cell deriverd ras Rars proteins 39i cteinS that bind and are in td r0 sine kinase re39c39eptor sig na1 transduction pathways in cells QRas proteins a Piase a ctiVity is in signal tranSdiJction is inactive in 3911 ff ah M o39st mutations that transfor1n ras into ran state inhibit its GTPrase activity thus it a perpetually on state for signal 11 Unt39t39l you h vc v hv lllll iuv Sramz A phage str ai n ca r rying an amber stop mutation in the S g ene involved in 39lysiS39 of the bavc39ter ial c4ell membriahej The Sam mutation is a suppressed by a suppress1 r lIN gere sup I9 ligate d t the genomic insert1 fh us allowinrg for ba39crterial cell lysis and plaqlle formation by recombi nant ph age Plate ibra on n Plaques only form Mich Includes an Li Q mmm 3T3 mom ce Transformed call foc grovvth properues oncogene Select cont ens wiih aifer 51 due to express39on of cans of su oci Transformed cell 1 Prepare DNA from primary ransfectant use in further transfeclion of 3T3 cells 2 Pick transformed eel foc g Second ry Kransfectant 1 Pre pa rc DNA and H Mouse DNA Pr re genomic DNA I brarv n phage lambda vector contain an amber mu In are l i one essential genes 19 2105 nsuppressing Eco139 host ed by phage Errylng DNA39 fragment sup F he ene and hence x oncog Transfect mouse eprt cells Sesec apn ceus P marv apn lxansfalztznt e it to transfect meuee apr ee 1 Prepare DNA from primeW apnquot transfectant and 2 Select apt t cells Secondary aprt transfectant prepare NA H Drzc H Prepare genomic DNA Ilbmry n phage lambda pla e out brary Screen plaques by hybrie panszz DNA as probe Isolate recumbinam phage carrying DBR322 and agociated eprz quot gene 39by quot 39 V to the isolatlorn of the I 39 u run 2 l 39 11 gene Polymorphic Sequences39 HPS OHPS c an be species and often individual specific OParticularly useful ide ntifying the origin of DNAS from higher eukaryotes vertebrates 7 lrants Examples of HP SL Re petitive Mieroqsatellite QSe uence Tawwed Sites SrliS39 0 L40 11 g Int e r sjpetrs ed N u lc39ea r Eleng ents 11 s39 QShort I39ntersp39enSed Nuclefar Elements SINES Alu 39S39equ39enc efs Digest DNA Circularize PCR r 7 CYCLE 1 PRODUCTS J 39 50 cycles 0 PCR 39 2 MAJOR PROBL CT Tar g eotxed vs Int e grrati on of Transgenes in Sells 39l ransgenes in Nlammalian Cells Transfected DNA is randomly integyrated in the host cell genome Homologouis Re39cornbina tion in cell s s ti1nuvlated by frere ends Freque ncy Sp n t2neous ree1 nb ina tion is 10W the rang ef of 103 to 10 tran s fe cted cell OPos39itiveNegative 39S elesction for isolatinrg transffected tnammalian cell s containing everits Dr MarioCapecohi University of Utah H A Swamims Updated 345 am PT Oct 8 2007 Three share 2007 Nobel Prize in medicine 2Americans 1 Briton honored fortechnique to manipulate mouse genes Mario Capecchi and Oliver Smithies and Briton Martin Evans were awarded the prize for deveIOping a technology known as gene targeting Gene Targeting Strategy of Mansour et al M Capecchi 1988 OTransfect Mammalian Cells with a quotReplacement Vectorquot containing a transgene anked by the genomic DNA sequences you wish to target plus a oneo gene which confers G418 resistance and acts as a positive selectable marker for DNA integration 01k of herpes simplex virus HSVtn wulCu in conjunction with gancyclovir acts as a negative selectable marker to eliminate transfections that have integrated into the DNA at random genomic sites Gancyclowr OH Gancyclov1r1s a nucleos1de analogue N which selectively kills cells expressing N t gt HSV tk because the substrate requirement HIINJN N of HSVtk is less stringent than that of L0 OH endogenous cellular tk OH Enrichment of Transfected Cells for Targeted Events a Positive I negative selection for targeted integration events Genomic DNA sequences you wish to target X HSV TK 7 G418 Positive Selection 7 7 Random integration via vector ends Hsv TK jL Cells G148 resistant Neo gancyclovir sensitive HSV TK Enrichment of Transfected Cells for Targeted Events b Negative selection for targeted integration events Gancyclovir 98 9 Ir mZ Z39 J Targeted integration via homologous integration 7 L Cells G148 resistant Neo gancyclovir resistant HSV TK39 Gancyclovir selection increases targetedrandom integrative events Targeted Gene Disruption Strategy in which Gene A in the Replacement Vector Target Gene has been Disrupted by Insertion of a neo gene HSV k I mgmle dldl1ri0l11 11 by vuluc oi rccomlmmgmm JCIIOn 11 DNA unrlc 1710 HSVrlk Transmmam Is Gala reslslzml 11mm 1 i A replacement vccmr luv mv q 39A39 m an n A has nua lyy n of a n 0 Ivnin mm whmh can 5 a llnkrul Hsv Ik q cm nno olnv G418 Gancylovir Two llonluluqous IEcuuvhumvmn CVI HIb in x hmwouu ymxc A scqw u u n cu mu lost a HSV u um HSVrlk s incnlpormed mm genuine lending m m u sunwwuy Translccmul s GMB resishml ancyclowr Ioslslnnl nea Genomic target gene A is disrupted by neo insertion Ir Speci c alrly Created Mammalian Cells With sites at Lovcat iotis in the Genomre O p riate C Ta rge ti 11g V e ct orrs 39 13 la s mid s o 1quot Vil ll39S e s A model of Cre Function incised from chrom Danme 3 r1 C I1 31 Tlh39e b ac t eriopha g e P17 acre g einie snort Ior y cl izainn icombin ation e n cordes a site spe cififc recombrignas e l ogically 139 E that reC ognizes piai red 34 nt lax P sites and d ele te39s the intervening between them Fukushi a S and B Sauer 1992 Proc Natl Acad Sci USA 89 7905 7909 oCre also promotes integrative recombination in eukaryotic cells containing a LoxP target site 7 reviousli introduced b7 transfe ction into the cells A single c0py or an inactive Lon neo fusion gene lacking a functiongall promo terr is integrated by selection into host crells to act as a 1390 c39hromos omal targre t C traensfectin f these cells With a quot39l oxlt recOmb inati on Vtaer get39ing rvect rquot c ontainjing a pzrotrioter ATG lox targeting Seq1391evnc39e a se conVcl expression Vect containinig the and skelemct for cell ulvar re sistance to the neomycin analg G418 O fiareign gene carried on the targeting vectorr heg a lacZ g ene Will also be irnrteg rate cl at the genro39l n igc P site OMethod is 95 0 ef cient Activation of a functional loxneo gene by sitespecific targeting pBS226 onP neo An DHFR J Cre CMV loxP neo An DHFR 39IIIIII OX g S39ccu39rc TCG 1m ACT rcc mr me ATA CAT TAT ACG MG TTA TAT START MG GGT TCC GOG CTR CC39I CTR GAG GTA CCT GGA TTG CAC 39 Tranasfe ctioll Techniques Jsed to Analyze Gene SV Rous Sarcoma Virus RSV Cytomegaloviurs CMV ii ContStitutiVe PromtersEnhance1 s usually frOm 39 39 hou se kerelai ng gene 39 39 acti39n tubulin cyto Chromeec ii Cell Tissuer ProAmoters insulinr promoter promoter cyclin AB ge ne promoters v Inducible P romot39ersEnhance rs H eat P39rO motje rs HS P eg induce d by thermal shO39C k and other stress factors Metall othio nine Prmote39rs induC39ed by metals eg z irlc5 cob39alt etc Promoter Analyses Selection of Reporter Genes i n HIT 1000 30 Typically want a heterologous reporter Should be easily measurable Soluble assays grind up the cells Luciferase Bgalactosidase Chloramphenicol Acetyl Transferase Secreted reporters evaluate media rwhHrmn Secreted Alkaline Phosphatase Staining Assays look at the cells directly Bgalactosidase Green Fluorescent Protein Should not be regulated posttranslationally OMammalian cells have little if any endogenous CAT enzyme activities B oth radi39ola ctiVe n onL radi oactive crhl orampllenieol substrates avaiglab le Dikre ctio n of Migration Quantitative Linear over a Wide cochentration range of enzyme VOQ uicl and easy to us More sensitive than the system Special Instr umen t lumin ometer requi recl Cells trans fecjted with plasmids contarining the luciferaSe gene are lysed to release rotein substrate luciferin added to the l sate in a I The catalyzes a rapid ATP dependent QXidation of the substrate Which emits light ORenilla synthetic lucifeliafsrej r sensitive39 an Quantitative Simple immunological detection procedure ELISA Product of transgene the 191 amino acid hGH peptide is secreted into the medium Convenient for use as quottransfecti on ef ciency standardquot When o tran f t ith other plasmid expression vectors 9 Green Fluorescent Protein GFP E Xtremely sensitive anrd ve rsatile O GFP can be made as a chimeric fufsfion feportef r protein OF lruorimeter re urired for detect139nr O Qualtntit atiVe a Wide concentrations O39I39n vivo c time measurements of GFP protein c an be made QSynthetic versiolns now come in 39red blue oth er cololrs 73 r1 3 741 2574 57 343 v 19 q 7 7quot 9 n l J1 E l LUUJGU A 7 in Question Is the promoter for the Scalyskin ssk gene regulated by cAMP ie does it contain a cAMP Response Element tILZE ToolsReagents 0 Cells that express the Scalyskin gene skin 0 Plasmids Vectors containing promoters linked to a measurable reporter luciferase o Scalyskin promoter sskluc How much rgt Positive control iiTEFIEETKluc 6 Negative control lcJE3TKluc o Promoterless control luc o Transfection efficienc control CMVBval o A transfection vehicle lipid mediated Introduce into cells Transfection Assa activit of re otter enes r eg luciferase Minimal PromotersResponse Elements Promoter Example Theoretical Results V6hCe CAMP 000E Lucnerase ActIVItyBgal ActIVIty Contains CRE Sites Protein Analyses Promoter Selection Promoter that works in cells of choice Inducible vs Constitutive Approach depends on question being asked Overexpression More activity than usual Misexpression Activity at the wrong time or place Reduced Expression Loss of activity Antisense siRNA Dominant Negative Protein Expression of a Mutant Altered activity in pie Question Does overexpression of Scalyskin induce proliferation ToolsReagents Cells that should respond to Scalyskin overexpression skin Plasmid capable of overexpressing Scalyskin Negative control empty vector Positive control vector encoding something known to induce proliferation in skin cells Proliferation assay Transfection approach stable vs transient transfection Assay to quantify overexpression Assessment of Protein FunctionVectors y EIRY AW Izmpty Vector 39 W Exp ressron 39 Vector V MV Antisense Vector Protein Analyses Example Theoretical Results p W 0 50 100 Proliferating Cells A 39vvv Ar3939 AVAuww AA 39 quot Internal Control for Transfection Ef ciency Transfm nquot Ef ciency Reference Plasmiu OIf reporter plasmid is Luc use Bgal CAT hGH GFP etc OTwo different assays using different reaction conditions o DualLuciferase Assays Fire y luciferase as a reporter and Renilla luciferase as a transfection ef ciency standard Promega Clone promoter of interest in front of firefly luciferase and use Rerillla iucuferase as an internal control cotransfect and assay Renlila luuferase driven by constitutive oter Effect of inhibitorsactivators on CRT promoterdriven luciferase expression 0 3 O at 03 gt 159 ugtlt 0 ethods for Analyzing PromoterEnhancer Regions Using Transfeetion Techniques Which sequences are involved in regulation elements Mann 139 anInIinIIinn anahlcic Gel retardation assays TRANS acting factors eg specific transcription factors rnninnc nf a nrnmninr oDltr in fth rmtrn activity quotgestion of that region by DNase I An endlabelled DNA probe is incubated with a protein extract or a gtI gtI ified DNAbinding factor The unprotected DNA is then Denaturing PAGE partially digested with DNaseI such is cut once Digestion products are then resolved ILALA Comparison of the DNase I digestion pattern in the presence and absence all nuAIAn II alln Ikn identification of a footprint protected region uonei gw o uogpaiga Foo rint quotAI quotLalt Electro Mobility Shift Assay EMSA N0 protein add protein Band Shift ncuDatIng a puri ed protein or a complex mixture of proteins eg nuclear or cell extract with a 32P Nondenaturing PAGE endlabelled DNA fragment containing the putative protein binding site from promoter region Reaction roducts are then anal sed on a nondenaturing polyacrylamide Retarded gel mobility clue to protein The specquCIty of the DNAbinding binding nr nlinnnunlnnlirlnc nalair an a binding site for the protein of F ee DNA b Interest or other unrelated DNA r pro e sequences VS No protein add protein Can use antibodies In con rmatory E L IEI LI LI mobility even further supershifts Antibody Against Protein Supershift Retarded mobility due to protein binding 2 tn 0 h 0 o O h u 2 LIJ O 9 u o 39 D E regulalcw protein A gence Vlilng I t l 39 I3 09 regIJaory pro Elf I ge z 39 lCROSSLINK PROTEINS TO Chromatln 1mmun0 rec1 1tat10n lDNA WITH FORMALDEHYDE LYSE CELLS ChIP SHEA K DNA INTO SMALL I 300 NUCLEOTIDEI FRAGMENTS g can 2 E An assay for interaction many Omar DNA fragmems that I I comprise the rest of the g of protelns Wlth PRECIPITATE DNA USIN ANTI R G BODIES AGAINST GENE EGULATORV PROTEIN A lb5u1atul sequences in vivo MALDEHYDE ROSSLINKS REVERSE FOR C I EMOVE PROTEIN cm AMPLIFY THE PRECIPITATED DNA BY PCR DNA REPRESENTING POSITIONS IN T E EN M T ERE OCCUPIEDVBVEENE BEGULAIORV PROTEIN39A IN THE ORIGINAL CELLS Figure 7732 Molecular Biology of the CeIl 4th Edition not just act as reporters of gene activation Rhian Morgan Department of Medical Biochemistry UWCM Transfection Techniques Increaucu wee yxp euuon vain of Function 39 D39ecreasrcu ucne LAyression Loss 39of Function Gene expression modulation agents delivery increase expression gain Of funcuon decrease expression loss of function increase expression Eukaryoticviral vectors Distal Promoter C D N A Intron m AAAA Temporal and spatial expression 1 Tissue speci c brain liver muscle 2 Ubiquitous actin housekeeping 3 Inducible tetracycline interferon Gene of interest C 11 1 Virus e s anlma s l Viral genome Decrease expression Antisense 1 Small singlestrand DNA oligonucleotides 15 25 bases gttagccggggaagcaaat caaucggccccuucguuua 33333 1 RNAse degradation of the RNADNA double strand molecule 1 U8ulullUll UDlllS all Inducible Gene System An Example of Inducible Expression Tet Off Premise I can t get stable cell lines that express protein Deadly I think that ex ression of this I rotein is lethal Constitutive Promoter Promoter CMV Deaolll y No Cells An Example of Inducible Expression Tet Off Addition of Tetracycline Tet RActivator Repressed Promoter TET 00 L0 a 39 ee lly quotle0 Cells Decrease exlzression Uommant negative Shuzture cf o nyc MaxDNA Luau zinm HIIZ M utant M ax Doesn t Bind DNA but still Interacts with Mad and Myc Dominant Negative because It reduces the am aunt of functional Mad and Myc in Cells Huh 1th Nair am Burley Cell um Decrease expression Ribozyme Transfect Cells with a Ribozyme Expression Vector CDNA l N N ribozyme uaagc ggcaaau auucg ccguuua aaaaa Cleavage of the target mRNA Decrease expression AntiS I lS 2 Synthesis of an antisense RNA complementary to the target I 9 a a CDNA L L L I I I 9 Target 5 T b T aaaaaa3 mRNA 1 l RNAse degradation of the RNARNA double strand molecule Decrease expression recombinat i011 Site speci c recombination Lox system Flp recombination From P1 phage Excise and integrate DNA Cre recombinase Lox site approx 30 bp EXCise Integrate Crelox Regulation of a Protein Expression r s L 173 p mi r I p I39 1 Tr r t D r Ubiquitous promoter Ubiqurt us promoter Short coding sequence i LoxP altn LoxP S L k Protein produced hereafter The gene lacZ has LoxP sequences containing a stop signal that prevent the gene from being ex ressed When ex osed to the Cre protein the LoxP and sto signal are excised and the gene is expressed Conditions in which the cre is present thus regulated the expression of the lacZ gene Decrease expression site speci c recombination CreZox floxed gene GDNTA K0 in the brain only KO iniheaduiomy E Temporal and Spatial Gene Inhibition via Gene Knockout SPECIAL ALL OTHER CELL TYPE CELL T fPES Cre0x Mouse Breeding Mice With the Cre protein expressing in a speci c cell type are bred With mice that contain a target gene surrounded by loxP sites When the mice are bred the cells carrying Cre will cause those cells to lose the target gene Nomenclature miRNA micro RNA stRNA small temporal RNA involved in developmental timing miRNAs siRNA small interfering RNA ncRNA noncoding RNA Riboregulators PTGS post transcriptional gene silencing What is RNA Interference RNA interference RNAi A phenomenon in which the introduction of double stranded RNA dsRNA into a diverse range of organisms and cell types causes degradation of the complementary mRNA A tool used to study the following Determine gene function Study pathways Identify and validate targets Generate knockout models Fa sf Transcriptimnl gem silencing PTESJ RNA interfmn e BINAij RNA silencing Han EH 223am Ilsa 231659 199D Cellular role of siRNA May serve as an antiviral tool Or protection against transposable elements Seems related to transcriptional silencing by induction of heterochromatin plants and yeast Biochemical Mechanism of RNAi imutuum4 mum M dsRNA is introduced into the cell DICER digests dsRNA into 21bp ds RNA short interfering Ms siRNAs The siRNAs undergo strand separation The siRNAs are integrated into the RNA Induced Silencing Complex RISC The antisense strand then binds to its compiem entarytarget m RNA Nucleases Within the RISC degrade the targeted m RNA Decrease expression RNAi siRNA caaucggccccuucguuugnn aaaaa TargetmRNA a Synthesis guuagccggggaagcaaan jauuugcuuccccggcuaac RNAi 30 to 50 nucleotides b cDNA gt 4 1 a l n Processed by the R Ai cell machinery dicer NA RNAse degradation Cleavage of the target mRNA le uestions Transfections oYou have a gene that is only expressed in breast cancer and think that it is regulated by estrogen 10 you test this 0 iv W ant t determine if Protein X reg u lates the for ge n e39 Y do test this O39You bie39lieve that Gerne is a tum or supp lfessor gerne ie pitreV39ents 39cavncerou s transforngtion f ete39l l39si test this b e ujse39d tO39 p otenti ally correct certa in 1391 disei39asegs
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