Red/ET Recombination

Hox-A11 homology region

ORI amp

minimal vector Hox-A11 homology region

Red/ET+ pRedET Recombination

pSub-Hox-A11 (18 kb) Cloning Without Restriction Enzymes

+ pRedET

pSub-Hox-A11 loxPneo loxP (19.5 kb) neo gb2 PGK loxP

+ pCre

pSub-Hox-A11 loxP (18 kb) loxP

+ pRedET A Guide to Next Generation Cloning

Hox-A11 Conditional knockout targeting vector loxP (19.5 kb)

loxP neo gb2 PGK loxP TECHNOLOGY with Red/ETRecombination Precise, High-SpeedCloning 3. Products 2. Applications 1. Technology Inside thisguide • • E. coli Animal Targeting Constructs Strain Optimization

......

. .

8 5 3 protein pairs, eitherRecE/RecTorRed E. coli in DNA of engineering the permits Recombination Red/ET at EMBLHeidelberg. developed technology ‘recombineering‘ patented the tion, engineering company to commercialise Red/ET Recombina Bridges Gene Recombination. Red/ET of applications the of overview an provides chure bro This products. and services licenses, recombineering Fromheadquartersits Heidelberg,in providesBridges Gene search. re your do to time you give to methodologies DNA tional conven alongside deployed be easily can Recombineering sential componentofthemolecularbiologicaltoolkit. freedom than any other technology and has become an es engineering DNA more permits therefore Recombineering neered atanysiteusingRed/ET. Any DNA molecule in restriction enzymes, PCR,DNAligase,becauseitis with pasting and cutting as such technologies, gineering en DNA other from differs Red/ET with Recombineering

• • Promoter independentofrestriction sites notlimitedbyDNAsize using homologous recombination mediated by phage a fudd n 00 s seils DNA specialist a as 2000 in founded was E. coli of almost any size can be engi

pBAD a /Red b .

------

TECHNOLOGY TECHNOLOGY

TECHNOLOGY Promoter

pBAD With Red/ETyoucaneasilymodifythe constructs for animal models: Red/ET allows for the genetic engineering of tailor-made targeting Animal targeting constructs molecules. DNA large of engineering the for applicable highly is it steps, up or reactions ligation enzymes, restriction on pend vivo in Red/ET exploits phage conventional cloningmethods. reliable modification of plasmids, BACs, or the highly and flexible more faster, a allows Recombination Red/ET DNA engineering? Why isRed/ETRecombinationasuperiorapproach to • • • • • • • • Germany perform your project. usingGene Bridges‘ kits or let our service facility in Heidelberg, Youcan establish Red/ET Recombination inyour laboratory by introduction ofpointmutations promoter finetuning reporter geneandtagintegration genedisruption,deletionorinsertion introduction ofpointmutations exonswapping promoter orreporter fusions conditionalknock-out/knock-in genetic engineering in engineering genetic strain modification l homologous recombination potential for E. coli E. . Since Red/ET does not de not does Red/ET Since . E. coli loxP/FRT PGK genome: E. coli gb2 in vitro in Selection Marker genome than clean- loxP/FRT Red/ET Recombination - S aet o. ,5,1 and 6,355,412 Nos. Patent US Red/ET Recombination Principle patentscovering Homologous Recombination. rected Cloning and Sub-cloning Using Di For Compositions And Methods U.S. Patent Application No. 09/350,830, Novel DNACloningMethod(ET). PCT/EP98/07945, Application Patent Japanese Patent No.EP4139561 European Patent No.EP1034260 B1 6,509,156B byStewart Red/ET ataglance • • • • • • cloningofinsertsupto80kb noligationreactions precise atany position sequenceindependent sufficient for recombination cloning without restriction enzymes sequence flanking of bp 50 only et al . -

TECHNOLOGY TECHNOLOGY TECHNOLOGY Three SimpleStepswithRed/ET The arms. homology by flanked quences se DNA of exchange precise base te promo pRed/ET plasmid from genes express which Cells Three simplesteps Biotechnology recombinationgous in homolocloningbyStewartA.F. DNA and J.P.P.,G.Muyrers Y.,TestaZhang 27 (1999) 1555-1557. ET-recombination. by chromosomes artificial bacterial of modification Rapid A.F. Stewart, G., Testa, Y., Zhang, J.P.P., Muyrers, 128. coli engineeringusing recombination in DNAStewart forlogicA.F.andnew A J.P.P. Muyrers F., Buchholz Y., Zhang Literature be reduced to three basicsteps: can tasks demanding most the Even annealing protein Red Red exonuclease the 3. Selection/Screening 2. Recombineering 1. AttachmentofHomologyArms . n vivo in NatureGenetics ecin s aaye by catalyzed is reaction 18 (2000) 1314-1317. Nucleic Acids Res. a 20 (1998)20 123- b l and the DNA the and E. coli E. . -derived . Nature red E. - - -

expressing plasmid. into introduced is insert The 2. Recombineering arms foranygivenlocuscaneasilybeattachedbyPCR. homology Thus,DNA bp. 50 only stretchesof homology terminal by flanked is which DNA linear requires Recombination Red/ET 1. Attachmentofhomologyarms Cells harboringtherecombinant DNAare selected. 3. Selection/screening Replicon Replicon E. coli E. pRed/ET cells propagating the Red/ET the propagating cells

50bp homologyarms Heterologous DNA recombination Red/ET mediated TECHNOLOGY APPLICATIONS Short Arm Mouse loxP ecombination r Short arm sm ES cell loxP FRT gb2 PGK loxP Long Arm Selection Marker gb2 - - - - - PGK FRT Animal Targeting Constructs Targeting Animal DNA modifications irre Exon in vivo loxP S ecombination r Long arm L ES cell Full documentation Steps to improve efficiency for subsequent ES cellrecombina tion or blastocyte microinjection Assistance with project design and verification strategy design and verification Assistance with project

Basic targeting construct Basic targeting Constructs can be prepared as high-copy plasmids with an overall an with overall plasmids as high-copy can be prepared Constructs kb. 50 to up of size a with plasmids low-copy or kb 30 to up of size • Size of the constructs • Our services include • why not take advantage of our service facility in Heidelberg, Ger Heidelberg, in facility service our of advantage take not why many? Use the modular system of our recombination kits and functional functional and kits recombination our of system modular the Use Alternatively, constructs. targeting optimized prepare to cassettes nation kits open up exciting possibilities for the fast and reliable constructs. engineering of targeting of developmental biology and modelling genetic disorders. and modelling genetic disorders. of developmental biology Red/ET Recombination enables spective of composition and size. Thus, Gene Bridges´ recombi and academic clients for tailor-made targeting constructs. constructs. for tailor-made targeting and academic clients technologies Transgenic are an essential component in the study At the cutting edge of DNA engineering, Gene Bridges‘ kits and cloning services fulfill theneeds of our pharmaceutical, biotech, How can Red/ET Recombination help you to make tai make to you help Recombination Red/ET can How models? constructs for animal lored arm). Appropriate restriction sites for linearization (L) and screening (S) can easily be incorporated. be easily can (S) screening and (L) linearization for sites restriction Appropriate arm). For For efficientES cell recombination, flanking homology arms can be extended to kb 5 (short arm) and10 kb (long APPLICATIONS MF APPLICATIONS Short arm Short arm Short arm FRT loxP Exon FRT FRT neo neo neo gb2 gb2 gb2 PGK FRT PGK FRT PGK FRT cDNA Exon Reporter P loxP P Long arm Long arm Long arm Promoter fusion constructs Promoter Fuse your cDNA to the of promoter without interest, introducing pattern. the expression additional nucleotides which may affect Conditional knock-out targeting constructs targeting Conditional knock-out Analyse gene function by flanking an essential exon with loxP recombinase. excision by Cre sites for subsequent Reporter constructs Analyse gene expression by a reporter gene fused to the native promotor without introducing additional nucleotides which may pattern. affect the expression - - - - - strategy and strategy glycerol stock E. coli in silico [email protected] +49 6221 13708 11 13708 +49 6221

Provide us with the gene name (NCBI name gene the with us Provide geting construct by sequencing. You receive an harboring the construct and a de tailed report. bone, providing large (>5kb) mology ho arms for an efficient ES cell recombination. Constructs ≥20kb are available as low copy plasmids upon request. tar final the of integrity confirm We for cross checking. for cross the BAC order appropriate clone We C57/BL6 strains mouse the on based or 129Sv. We clone ≤18kb of the modified al lele into a high-copy vector back Acc. No.) and kind of modification. Acc. No.) and kind develop an We data electronic the with you provide

Gene Bridges contact: 1. Custom service work flow for Custom service constructs targeting 6. 5. 3. 4. 2.

Animal Targeting Constructs Targeting Animal

APPLICATIONS APPLICATIONS APPLICATIONS Short arm Short arm Animal Targeting Constructs Targeting Animal Exon plasmid Low-copy Murine Exon Exon HumanExon Gene of interest Exon BAC clone BAC Murine Exon Long arm Long arm

exon boundaries are preserved in the transgene construct. in the transgene preserved exon boundaries are Transgenes Transgenes are generally more reliably expressed if the intron- a low copy plasmid. Unwanted flanking sequences areremoved yielding optimized constructs. Optimized transgene constructs Optimized transgene into clone BAC a from kb 50 to up locus genomic whole a Transfer Replace a given exon with the human counterpart to study the allele. influence of your drug on a human-derived Targeting constructs to humanize animal models Targeting base pair mutations at any position. base pair mutations Targeting constructs introducing point mutations constructs introducing Targeting Study the effects of SNPs in your animal model and insert single

APPLICATIONS APPLICATIONS APPLICATIONS - Genome of marker eening for E. coli r FRT/loxP Sc loss Enter another modiﬁcation cycle if appropriate strains or use our service genes E. coli FRT/loxP Genome sm sm FRT/loxP E. coli FRT/loxP is frequently used as model organism and functions functions and organism model as used frequently is emoval FRT/loxP r ecombination strains? Genome r e r Red/ET recombination E. coli Gene disruption, deletion, insertion, modification Gene disruption, deletion, integration Reporter gene or tag fine tuning Promoter nome modifications can be achieved. nome modifications can be achieved. How can Red/ET Recombination help you to optimize coli Escherichia as microbial factory in Recombination with biotechnology. Red/ET modifications: chromosomal to access rapid and defined a enables • • • Usethemodular system ourofrecombination kitsand functional cassettes to prepare optimized facility in Heidelberg, Germany. Markerless knock-out of In combination with Flp/Cre technology multiple markerless ge Selection marker by Flp/C FRT/loxP - - - strain*. E. coli glycerol stock glycerol 08 11 E. coli E. 1 137 strains can be provided by Strain Modifications Strain E. coli strain optimization strain [email protected] +49 622

You receive an receive You and a detailed report. Provide Provide us with a reference se tronic data for cross checking. data for cross tronic We confirm clone integrity by se quencing. quence file and your quence file and your design and provide you with elec We will help to optimize the project project the optimize to help will We

*common Gene Bridges. Gene Bridges contact: 1. Custom service work flowfor 3. 4. 2.

APPLICATIONS APPLICATIONS APPLICATIONS - - - - Transfor , encoding A. under cont cfp Fluorescence

A. with Strength Promoter pgi-lacZ B B cells achieved by chro by achieved cells GeneX E. coli E. Promoter activity of a subset B. Dark field microscopy. Pictures B. A of SPL clone. Red: native promoter. of SPL clone. Red: native promoter. 45, No 3, 2008). Vol (Biotechniques, Protein tagging in tagging Protein mosomal fusion of for cyan fluorescentprotein. microscopy. of Institute Weizmann Lab, Stavans by provided Science, Israel. Analysis of the activity of rol of a synthetic promoter library. mants appear white to dark blue in the pres ence of X-gal. A - - Strain Modifications Strain ATG -sm). FRT rpsL ATTGCTA sm Gene X FRT GAAGAGT SPL AAATCA 5 Reporter FRT sm TATAATN sm 17 Gene X FRT rpsL TTGACAN 5 N

Optimize gene expression by fusion of a synthetic promoter li (SPL) to the gene of interest. brary Promoter fine tuning Promoter Use a chromosomal reporter strain to analyse single copy gene expression. Reporter gene or tag integration Insert point mutations or other seamless modifications by em cassette ( - counterselection ploying a selection Seamless modifications

APPLICATIONS APPLICATIONS APPLICATIONS MF - - - - sm Plasmid ori sm BAC pRed/ET Plasmid ori Flanking of the cloned fragment with functional cassettes or restriction sites easily possible Fast and simple cloning of large fragments Fast and simple cloning of large plasmids Cloning size up to 30 kb for high-copy plasmids Cloning size up to 80 kb for low-copy sites Cloning independent of restriction • A plasmid backbone which contains an origin of replication (ori) and a selectable marker (sm) is PCR-amplified withprimers intro ducing homology arms (red). How can you take advantage of large BAC libraries? advantage of large How can you take cur are organisms eukaryotic 400 than more for projects Genome projects, these of majority the For finished. or running either rently librar chromosome) artificial (bacterial BAC insert large annotated available. ies are accessible. easily source valuable this makes Recombination Red/ET plas into cloned be can sequence any of fragments genomic Large mids by gap repair. The method is not restrained by the general of PCR. fidelity and amplicon size limitations Advantages of BAC subcloning • • • • http://www.genome.arizona.edu CUGI Institute Genomics University Clemson http://www.genome.clemson.edu Geneservice http://www.geneservice.com AGI Arizona Genomics Institute BPRC Children‘s at Center Resource BACPAC Institute Hospital Oakland Research http://bacpac.chori.org Selected BAC resource centres resource BAC Selected

Large Fragment Cloning Fragment Large APPLICATIONS 10 PRODUCTS 11 www.genebridges.com [email protected] pRed/ET expression plasmid pRed/ET expression experiments Suitable control sequences Detailed manual, maps, Academic researchers can order Gene can order Academic researchers cassettes and plasmids Bridges kits, our website: from distributors. our regional or from Commercial organisations require a license from Gene Bridges to use the Red/ET Recombination Please contact: technology. • • • Kit Contents

- - rpsL E. coli chromo . R neo Red/ET Recombination Kits Recombination Red/ET E. coli R amp . R Cat.No. K001 fragments BAC of deletion for Developed and all types of Functional elements: Tn5- basic modifications. some. Functional elements: Linear vector Linear elements: Functional some. ColE1 ori- negative selection marker cassette neo Cat.No. K003 Developed to transfer fragments up to 30 kb from BACs or the Cat.No. K002 Developed for the insertion of mutations in point BAC clones or the genome. Functional elements: Positive/ Recombination Recombination Recombination Easy BAC Modification Kit Easy BAC Modification Version 2.6 (October 2007) Version 2.5 (December 2006) Version 3.0 (January 2007) Red®/ET® Red®/ET® Red®/ET® By Quick & Easy Modification Kit BAC BAC Subcloning Kit BAC By Counter-Selection Modification Kit BAC Modification Kit) BAC (Advanced By Technical Protocol Cat. No. K001 Technical Protocol Cat. No. K003 Cat. No. K002 Technical Protocol Quick BAC Subcloning Kit Counter Selection BAC Modification Kit Counter Selection BAC Modification

Establish Red/ET Recombination in your lab - Gene Bridges offers Establish Red/ET Recombination in your lab - Gene Bridges offers cassettes and plasmids. modular of kits, a wide range PRODUCTS MF PRODUCTS 1313 - - - - - E. Gene Deletion Kit genes. Markerless modifica Developed for the deletion of coli tion possible in combination with expression plasmid A104 or A105 (see page 14). Cat.No. K007 Developed to insert a SNAP-tag cassette into a BAC clone to con firm the specificity of an RNAi- based loss-of-function phenotype. Determine the trans (LOF) fection efficiency by the integra ted SNAP-tag casette. Cat.No. K004 (loxP), K005 (FRT) Cat.No. K004 (loxP), Developed for the insertion FRT of or loxP into respectively sites, ex Cre or Flp plasmids. high-copy plasmid included. pression Functional elements: loxP-PGK-gb2-neo-loxP (K004) (K005) FRT-PGK-gb2-neo-FRT Cat.No. K006 Version 1.1 (June 2007) Recombination & Version 1.3 (June 2007) Recombination Recombination Recombination Easy RNAi Rescue Kit Easy Easy Conditional Knock-Out Kit Easy Conditional Version 2.1 (November 2007) Version 1.2 (January 2007) Red®/ET® Gene Deletion Kit Quick & Easy Kit Knockout Conditional (loxP/Cre) By Red®/ET® Red®/ET® Red®/ET® Quick & Easy coli E. By Quick & Easy Rescue KitRNAi By DetectionTMR-Star SNAP-tag™ Quick & Easy Kit Knockout Conditional (FRT/FLPe) By Technical Protocol Cat. No. K005 Technical Protocol Cat. No. K006 Technical Protocol Cat. No. K007 Technical Protocol Cat. No. K004 Quick Quick Quick I I I I L S F T B C C A A A G U N N N D N N D D O O O M loxP loxP pCre pBAC pRedET pRedET pRedET Hox-A11 PGK - gb2 - neo PGK - gb2 - neo loxP loxP + + + + Knock-Out Kit (loxP/cre).

Hox-A11 homology region Hox-A11 p

p p loxP p p m m m a m m a a a a amp PGK loxP (18 kb) (18 kb) gb2 loxPneo Hox-A11 (19.5 kb) (19.5 kb) Hox-A11 pSub-Hox-A11 pSub-Hox-A11 pSub-Hox-A11 pSub-Hox-A11 pSub-Hox-A11

targeting vector targeting

neo

I I

I R

minimal vector R

O R

R O

loxP O

Conditional knockout O O loxP ORI PGK Easy Conditional gb2 neo loxP loxP loxP Hox-A11 homology region Hox-A11

5. Red/ET Recombination 4. Cre - mediated Excision 4. Cre 3. Red/ET Recombination 2. Red/ET Recombination 1. PCR Construction of vector a for conditional the knock-out targeting Kit Subcloning BAC the using Hox-A11 protein homeobox murine and the Quick Constructing conditional knock-outs Constructing conditional Red/ET Recombination Kits Recombination Red/ET

PRODUCTS PRODUCTS 1212 PRODUCTS 1313 Eukaryotic promoter promoter Prokaryotic site target Flp recognition site target recognition Cre Neomycin Kanamycin Chloramphenicol Ampicillin Hygromycin : : : : : : : : : PGK gb2 FRT loxP neo kan cm amp hyg

R R R R R R R - - - - hyg hyg neo neo neo neo neo Selection Selection Cassettes Selection Mammalian - - - T7 R R R R R R R R R R R cm cm kan kan kan kan hyg hyg kan amp amp pA Selection loxP/FRT sm gb2 loxP-gb2-amp-loxP FRT-PGK-gb2-hygro-FRT loxP-PGK-gb2-hygro-loxP FRT-gb2-cm-FRT loxP-gb2-cm-loxP FRT-gb2-amp-FRT loxP-PGK-gb2-neo-loxP FRT-PGK-gb2-neo-FRT-loxP loxP-FRT-PGK-gb2-neo-FRT Cassette PGK-gb2-neo FRT-PGK-gb2-neo-FRT PGK overview loxP/FRT A011 A010 A007 A001 A002 A009 A006 A008 A003 A004 A005

Zero background because cassettes are encoded by suicide plas suicide by encoded are cassettes because background Zero clones positive false avoid to mids in a Cre or Flp recombination step where appropriate step where or Flp recombination in a Cre Due to selection a cassettes modular can architecture, be PCR- amplified with master primers otic (gb2) promoters, respectively otic (gb2) promoters, Selection markers flanked by loxP or FRT-sites can beremoved Selection cassettes are driven by eukaryotic (PGK) and prokary and (PGK) eukaryotic by driven are cassettes Selection Cat.No.

T3 Cassettes Basic functional cassette • • • Cassette Features • Increase your Increase flexibility by using additional selection marker cas Red/ET recombination settes optimized for

PRODUCTS PRODUCTS 1212 PRODUCTS MF R R R R R R , amp , amp R R tet tet cm cm Selection Selection puro puro 705-Cre 706-Cre 707-FLPe 708-FLPe Nature Biotechnology, 16:657-662 (1998)). Biotechnology, Nature Nature Biotechnology, 16:657-662 (1998)). Biotechnology, Nature et al. et al. Plasmid / Recombinase Plasmid (Academia) pCAGGS-FLPe (Industry) pCAGGS-FLPe Plasmid / Recombinase Plasmid Puromycin Eukaryotic promoter promoter Prokaryotic : : : A112 A113 A105 A201 A104 A202 Cat.No. Cat.No. For the removal of FRT-flanked DNA, e.g. neomycinresistance in mammalian cells. markers For the removal of FRT or loxP flanked DNA of FRT For the removal controlled tightly are propagation plasmid and expression Gene by temperature markers antibiotic resistance Available with various pBR322 or pBS pUC, (e.g. plasmids based ColE1 with Compatible derivative, RK2, R6K, cosmid, P1 and BAC)

Eukaryotic expression plasmid for FLPe Eukaryotic expression • *Flpe is a thermostable more derivative of wild type Flp displaying an enhanced activity at 37°C (Buchholz pCAGGS- the of conditions and terms the by governed is product this of use The Agreement. Material Transfer FLPe puro CAGGS CI-578 Optimized Cre and FLPe* expression plasmids for an efficient site- efficient an for plasmids expression FLPe* and Cre Optimized recombination. specific and Cre plasmids Flpe expression Prokaryotic • • • • *Flpe is a thermostable more derivative of wild type Flp displaying an enhanced activity at 37°C (Buchholz

ORI IRES puro pCAGGS-Flpe 705-/706-Cre 705-/706-Cre 707-/708-FLPe

Recombinase Expression Plasmids Plasmids Expression Recombinase PRODUCTS 14 FAX to Gene Bridges

Dear Customer,

for detailed information please use the form below and fax to: FAX: +49 (0)6221 13708 29

I am interested in:

Animal Targeting Constructs E.coli Modification

Gene Bridges Services Commercial licenses

My questions or remarks:

......

Name ......

Company/Institution ......

Building/Room ......

Street ...... City ......

State ...... Country ......

ZIP/Post Code ......

Telephone ...... E-Mail ......

MF www.genebridges.com www.genebridges.com 15 Gene Bridges GmbH Im Neuenheimer Feld 584 69120 Heidelberg Germany

Phone +49 (0)6221 13708 10 Fax +49 (0)6221 13708 29

E-Mail [email protected] Web www.genebridges.com

Court of Jurisdiction Amtgericht Mannheim, HRB 338013

VAT ID / Umsatzsteuer-Ident-Nr. DE 213116680

Authorised to Represent Gary Stevens, Youming Zhang, Francis Stewart