Lecture16 - wast voL 14 115—132(1993 Designer Deletion...

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Unformatted text preview: wast voL. 14: 115—132(1993} Designer Deletion Strains derived from Seeshoromyees eerevtstee 8288C: a Useful set of Strains and Plasmids for PCR-mediated Gene Disruption and Other Applications l[Cit-"iRRIlEi BAKER BRACHMANN, ADRIAN DARIES; GREGDRY .I. (DST; EMERITA CAPUTD, JDACHIM LIT, PHILIP HIEI'ER AND JEF D. EDEKE" Department of Mats-ruler Biology and Genetics, Jest-is Hepktns Untvsrstty School esttttctas, Baltimore, MD 21.295, LISA. Received 9 June 1997; accepted 13 June 199? A set of yeast strains based on Seeehammyrss esrsvtrtes 523 EC in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We aLso report the construction of new members of the pRErlilll series of vectors; mntaining the its-HM}; ADE! and METIJ' genes. (if: 1993 John Wiley 3: Sons, Ltd. Yeast l-Il: lIS—lfl; I995. mar wroaos— Saschammyrss esrsvtstes; yeast; gene disruption; 3238C; bacteria-yeast shuttle vectors; auxotrophic markers C. B. BRACI-IMANN ET A]... L Maw-W + right e la left righl an m an "9” Figure 2. it'Iethod of construefioo of designer deletion strains. An example is given in which LIFE?” is deleted to create {HEAL}. 'Ihe LYSE DEF is indicated by a white oral and the hatched regions indicate left and right flanking sequences. Lines with arrows indicate olignnucleotide primers for PCB-amplification of the flanking; regions, and the restriction sites engineered into these oligns are indicated by K, 33311; B, BmHI or 5, Sad. Using these oligos, the flanking regions are PER-amplified, out with the indicated enzymes and cloned into [IRS-106 to create a deleter plasmid; in this case, pADZ. FADE is then linearized at the unique Ciel {C} site in the right flanking region and transformed into yeast. Integration of the linearized yeotor within the genomic LIFE? right region rmults in the configuration oi‘seqoenees shown in the middle panel of the diagram. Recombination between the two left regions results in deletion ofthe LIFE? and 1rector sequences. Not shown, but equally likely, is recombination between the two right regions which results in restoration ofthe wild-type LITE” allele. DESIGNER DEIETION YEAST STRAINS AND NEW YEAST VECTORS _2256hn _134bp _4-53bn —2235bp v #— L YS2' area b 1621 m THPl‘ —- woe hp —HA3 -—I- _1112 hp J:mrrcrc> / _ RS— GEGCWHBTmGi-mcmmg Noi'el TAWm-raccocmaoaroc— P MEAD-EMMA? [blunt-J [FITWMMA‘PGGWE‘TMG— pRS- Selectable marker One-step gene replacement . Se : able marker Figure 8. A universal primer set for producing PER-mediated gene disruption cassettes. The double-stranded sequence shown represents about .50 bp surrounding a unique Ndel site in the parental plasmid backbone of all pRS-OUO series vectors [the parental backbone is a pBLUESCRIPTIpBLUESCRIBE hybrid made by ligating a PWI fragment of pBLUESCRIPT II—SK (containing the polylirrloer region] to a PvuI fragment of pBLUESCRIBE [containing unique NdeI and Aarll sites)]. Minimal DNA segments that encode each of the selectable markers (arrows indicate transcriptional orientation] shown above were blunt-end ligated into the blunted Ndel site for each pRS-llOD series vector. A single pair of oligos can therefore be used to amplify by PCR each of the selectable marker genes‘ Sequences of aligns used for amplification of the selectable marker genes are as indicated. Both oligos are drawn 5' to 3’. As indicated, 40 nts ot‘sequenoe from either the upstream or downstream flanking region ofthe gene to be deleted, in this case YFG, is added to the 5' end of each oligo. PER-amplification ofthe auxotrophic marker allele is performed using any pRS integrating vector as a template (this is possible because the oligos are specific to the sequenoejust upstream and downstream of the NdeI vector site into which all markers were introduced). The resulting double-stranded PCR product is then transformed into yeast, replacing the genomic YFG allele by a double cross-over event. The resulting disruptions contain any oil‘ eight selectable markers transcribed in the orientations indicated. Functional Characterization of the S. cerevisiae Genome by Gene Deletion and Parallel Analysis Elizabeth A. Winzeler." Daniel D. Shoemaker.“L Anna .lu.stromol"l‘.""I Hong Liang.“L Keith Anderson.‘ Bruno filndre.El Rhonda Bangham.ll Rocio Benito.5 jef D. Boeltef' Howard BusseyF Angela M. \iIhu.‘I Carla iIZonnellyr,a Karen Dawis,‘I Fred Dietrich,“ Sally Whelen Dcrlililr,z Mohamed El Buaid-roury.“ll Francoise Foury.“ Stephen H. Friend.2 Erik «Gentalenfi'iI Guri Giaeverf' Johannes H. He‘gemann.12 Ted Jones.‘I Michael Laub.‘I Hong Liao.4 Nicole Liebundguth.I David J. LocI‘thartJ‘I Anca Lucau-Danila," Marc Lussier,T Nasiha M'Rabet.3 Patrice Menard? Michael Mi'i.'tn'lann."'I lllhai lii'ai.‘I Corinne Rebischung.“ jose L Rewuelta.5 Linda Riles.‘IEI Christopher]. Roberts.2 Petra Ross-MacDonald.4 Bart Scherensf"|I Michael Snyder.“ Sharon Sooichai-Mahadeo.E Reginald K. Storms.T Steeve 1'uli'IEronneau..? Marleen Voet.” lliiuido Volckaert." Teresa R. ".iiiard.IE Robert Wysocki.” llirace S. "I"en..‘I Kexin "i'u..6 Katja Iimmermann,12 Peter Philippsen,“ Mark johnston.‘IEI Ronald W. Davis“? The functiora of many open reading frames [DRFs] identified in genome sequencing projects are unknown. New,wholevgenome approaches are required to systematically determine their function. A total of 5925 Saccharomyces cerevisiae strains were constructed. by a highvihroughput strategy. each with a precise deletion of one of 2026 DRFs [more than one-third of the DRFs in the genome]. Of the deleted DRFs. 1? percent were essential for viability in rid'I medium. The phenotypes of more than 500 deletion strains were assayed in parallel Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium. MW 3 MW»! 11 Hal-PW 2 12mm HT 13 W Wis-E unwirI-IH g 1-5 WWW WW' mflPHrir’rHr-i 5 unclear classification Ionic homeostasis _ Chromosome coll rescue, cell defence and cell “1113 Bison] transduction cellular Hoses-Ices} intracellular MW transport facilitation Fig. 1. {A} Genomic locations of 1620 nonessential {short black bars} and 3-56 asential genes [all black bars]. Deletions were generated in consecutive groups on multiple dirornosornfi. A lighter gray background 'ndifiitfi the location of chromosomal duplication blocks {23]. For 15 of the 355 essentid ORFs. a haploid null mutant. had been previously described. These inconsistencies may be due to differenca 'ru stra'n background or in the conditions used for germination of spores. For example. the previously-constmctedyhrlfllc [bigl] andyh' 196:: {pgil} null mutants show glucose sensitivity, and theyerSEc {”155} deletion rnulant requirfi a riboflavin supplement for viabl'rty 24}. In several cases haploid null mutants were reported to have slow— —growth phenotypes mr303c [Fifi], yolClZEC, ypi243c {9:063}. ypl'Ech [srpTEL and ydr353w [is-r1] {25}] or to be temperature sensitive [ydrl 13c {pdsl} andynglEc [gpil] [26]]. in some cases, haploid deleb'ons strains were constructed for genes previously determined to be essential by others [HKRL RNM. and Hi”? [EH]. [B] Distribution of functional classes of essential [inner circle] and nonessential {outer circle} OllFs using criteria from the Munich Information Centre for Protein Sequences [MIPS] {m}. Brain Struct Fun-at [2011]} 21491—109 DGI 10.1110?! sflfl429-m9-023fl-3 Animal transgenesis: an overview Miguel A. Gama Saaa * Rita Da Gaaperi * Gregory A. Elder Target Gene Hemete-geus tetra mbinatien Linearized Targeting Fetter l Geneticin {(3413} Selection Meeified Alleie Fig. 1 Hflmfllegeus reeemhinafien. In the example above the hemelegeua reeemhinaiien results in a gene l-meekeut cassette A cassette B ”Floxed” cassette B W _W ‘—* ‘9 l tamoxifen Y i inactive active Fig. 2 Tamoxifen-inducible conditional gene knockout. creEr chimeric (Ire-estrogen receptor protein. hsp heat-shock protein 90 Fig. 3 Schematic representation of Tet-regulated systems. Figure based on Stieger et al. (2009) 1. Basic Tet-regulated system cassette A cassette B mm a mum 2. TetOff dorinduced inactivation of tTA. tTA continuous expression A sum ~ m—_m 3. TetOn ' rtTA expression l—mmonl dos-induced activation of rtTA Knockout Rats via Embryo Mioroiniootion of Zinc-Finger Nuoloasos Aron lit. Geurts,"3‘r Gregory]. Eostf" ‘I'e'urgeniy Freylrertf‘ Bryan Ieitler,El Jeffrey C. Miller} ttitrian H. Ehoif Shirin S. Jenkins? Adam 't'd'oori,“| Iiaoxia Gui,“ Iiangdong Hengf' Anna Vincent} Stephen tam} Hieuyslarrr Juli-r:hallrienrirz,"2 Rebeeea Snohilling,"2 Jamie Foeeirlerf Shawn Hallo-nay} l-Iartmrrt 'tl'rl'eiler,1'2 S-éyerine Intrinoret,5 lgnaeio finegonf Gregory D. Davis,“ Lei Ilrangf Edward ]. Reharf Philip D. Gregory} Fyooor D. Llrnoyf Howard ]. lar:oh,"2"'1* Roland Buelorrth he hhmatny rat is a “ell-established “EdehmedfllmElFflshE-fihmhizygms am: nuielfisrmegmujedissaeficm thtllttlfll EFF-nangmiem inlaedSS 11hhl3;CiFPEZFMs]. strata- disassa-Ichhd hails {i} finite the fit 91 hllaed FI-II-I {Fem-headed hype-Busing III“ .. flutmgetedmdificafiencfflsgmcmeishrgely Rahfifl EFfls]. and 2193 :1de SD {Spleen range inhadahle. We hlvefljgsted the applicaaticm cf Dawley; Igtvt cIc'Ft-nls] flflbl'yflfi by French: 123' d:-..;.- A T"??? m“ EFF e—Kpresaian +— + + - - 35 NT lIFH-irhduzd cleavage 1mfl_ EDD- ‘l‘nepu tn.- HHEJ TUD- [1—51an Target! Enutil Injlct-d!“ Feundnra Era-d1 F1 Genus-Mt Elan r1 "'1'. 1 hhfl'l‘ll'tfl SE GFFIm RNA PH 1115' was {$3.5 5% {3:1} 2 [411} 11'1 5D lgMJ'Fiasmid PNLHD ensues [B1] 54111} M11} #3 an lgMJ'HaEI‘nid Flt-HE 5115MB :‘m Elana} Emu} NI] 5D lgMJ'Flasmid Pum4 511F423 1113i HHS} 4 {IE} MI] 51) Ighlmet-la. PM 1111] 1H1U4 [515] 14 [13} 41:29} N I] 5D IgmmRt-la. FHH? east-42 (E: 21 {15} 4 [19] HI] 5D IghtrmRt-ta. emu-1 13231115 {64] 19(16} 1 {5} HI] an Igwmnua Ica'tn 212119? :12] 4 {2; 3 [T5]: N D an Igurmnua Ice 15mm tfifl] 11"{13} 21:12} ND FHH 11mm RNA ICE 91.133 (91: #12131} 1 {E} N D PHI - pmnunlaar Injustice"; I1: -|nh'nr:1r'lnplalmi:; ' -ufl:ranal‘arrad; 1'- at Itvahuni found-urn; HI} - nut datamina-cl Fig. 1. IHnl-mediated gene disruptinn in rat emhrvus. ll} EFl'nls ccrntaining fine ctr Silt fingers were dfiigned ta target ceding sequencfi crf interest {grav ling} far site-specific cleavage. ll;L We at five pups burn after nricrninjectinn n1 EFF-targeted IFl'nls were devnid at EFF errprasicln. {C} Fuhrmerase chain reactiun using EFF-specific primers revealed truncated hut ncr wild-type sequence in each of the GFP negative pups campared with pnsitive Iittermatfi. 55 indicatfi Dahl 5 central DNA; HT indicatfi nn template. {IllL Table at injection data revealing succfiful muhgenais at the three gene targe1s after multiple delivery methods and doses in three rat strains. mgineued mange: mule-uses [EH-Is {2}] far mam: sgmmnfmmm flu- nI-imi-n-nl-im nFewifir-ret nun-ne- Fur-Mime amt-II fu' m'lifl'slfi. at rI-i-FHI-rmt mat-“heath“ Heinle- EH alarms ...
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