1、nAnimal model and functional genomicsnProteomicsnDrug leadsDNA microarraynGene expression analysis using a DNA microarray.The first step is to purify mRNA from each of the two populations that are to be compared,for example cells treated with a certain substance and untreated cells.The two different
2、 mRNA populations are used to prepare fluorescently labelled cDNA,targets,by a reverse transcription reaction in the presence of fluorescently labelled nucleotides.Two different fluorescent labels are used,one for each population.For example,the control mRNA population can be labelled with a red flu
3、orophore(fluor),and the treated population with a green.DNA microarrayThe two labelled cDNA pools are then mixed and hybridised onto the array.After hybridisation,the slide is washed to remove unspecifically bound cDNA transcripts.The slide is scanned with a fluorescence scanner,using different lase
4、r wavelengths to excite the different fluors,and the intensity of each colour in each spot is measured.From these measurements,a ratio between the two fluors is calculated to monitor the relative abundance of each RNA transcript in the two populations.These data are then interpreted with the help of
5、 various computer programs.Functional genomicsFunctional assessment of every gene in the mammalian cell is an on-going challenge.Understanding,not only the function of each gene in isolation but the complexity of functional networks and control systems is of particular importance for discovery of no
6、vel and valid drug targets.Answering complex biological questions in this context necessitates high-throughput gene functional characterization using an array of genomic,proteomic and in silico-based tools and technologies.Drug target discovery and validationnGene knockoutnGene traping and enhancer
7、trapingnRNAiTwo methods of producing transgenic mice are widely used:ntransforming embryonic stem cells(ES cells)growing in tissue culture with the desired DNA;ninjecting the desired gene into the pronucleus of a fertilized mouse egg.The Embryonic Stem Cell Method(Method 1)nEmbryonic stem cells(ES c
8、ells)are harvested from the inner cell mass(ICM)of mouse blastocysts.They can be grown in culture and retain their full potential to produce all the cells of the mature animal,including its gametes.The Embryonic Stem Cell Methodn1.Make your DNA Using recombinant DNA methods,build molecules of DNA co
9、ntaining the structural gene you desire(e.g.,the insulin gene)vector DNA to enable the molecules to be inserted into host DNA molecules promoter and enhancer sequences to enable the gene to be expressed by host cellsn2.Transform ES cells in culture Expose the cultured cells to the DNA so that some w
10、ill incorporate it.n3.Select for successfully transformed cells.The Embryonic Stem Cell Methodn4.Inject these cells into the inner cell mass(ICM)of mouse blastocysts.n5.Embryo transfer Prepare a pseudopregnant mouse(by mating a female mouse with a vasectomized male).The stimulus of mating elicits th
11、e hormonal changes needed to make her uterus receptive.Transfer the embryos into her uterus.Hope that they implant successfully and develop into healthy pups(no more than one-third will).The Embryonic Stem Cell Methodn6.Test her offspring Remove a small piece of tissue from the tail and examine its
12、DNA for the desired gene.No more than 1020%will have it,and they will be heterozygous for the gene.n7.Establish a transgenic strain Mate two heterozygous mice and screen their offspring for the 1:4 that will be homozygous for the transgene.Mating these will found the transgenic strain.The Pronucleus
13、 Method(Method 2)n1.Prepare your DNA as in Method 1n2.Transform fertilized eggsnHarvest freshly fertilized eggs before the sperm head has become a pronucleus.nInject the male pronucleus with your DNA.nWhen the pronuclei have fused to form the diploid zygote nucleus,allow the zygote to divide by mito
14、sis to form a 2-cell embryo.n3.Implant the embryos in a pseudopregnant foster mother and proceed as in Method 1.An Exampletargeted gene insertion requires the desired gene nneor,a gene that encodes an enzyme that inactivates the antibiotic neomycin and its relatives,like the drug G418,which is letha
15、l to mammalian cells;ntk,a gene that encodes thymidine kinase,an enzyme that phosphorylates the nucleoside analog gancyclovir.DNA polymerase fails to discriminate against the resulting nucleotide and inserts this nonfunctional nucleotide into freshly-replicating DNA.So ganciclovir kills cells that c
16、ontain the tk gene.gene knockout and animal modelnStep 1nTreat culture of ES cells with preparation of vector DNA.Results:n Most cells fail to take up the vector;these cells will be killed if exposed to G418.nIn a few cells:the vector is inserted randomly in the genome.In random insertion,the entire
17、 vector,including the tk gene,is inserted into host DNA.These cells are resistant to G418 but killed by gancyclovir.nIn still fewer cells:homologous recombination occurs.Stretches of DNA sequence in the vector find the homologous sequences in the host genome and the region between these homologous s
18、equences replaces the equivalent region in the host DNA.nStep 2nCulture the mixture of cells in medium containing both G418 and ganciclovir.nThe cells(the majority)that failed to take up the vector are killed by G418.nThe cells in which the vector was inserted randomly are killed by gancyclovir(beca
19、use they contain the tk gene).nThis leaves a population of cells transformed by homologous recombination(enriched several thousand fold).nStep 3nInject these into the inner cell mass of mouse blastocysts.nGene knockoutnProteomicsnDrug leadsKnockout Mice:What do they teach us?nIf the replacement gene
20、(A*in the diagram)is nonfunctional(a null allele),mating of the heterozygous transgenic mice will produce a strain of knockout mice homozygous for the nonfunctional gene(both copies of the gene at that locus have been knocked out).nKnockout mice are valuable tools for discovering the function(s)of g
21、enes for which mutant strains were not previously available.Knockout Mice:What do they teach us?nTwo generalizations have emerged from examining knockout mice:nKnockout mice are often surprisingly unaffected by their deficiency.Many genes turn out not to be indispensable.The mouse genome appears to
22、have sufficient redundancy to compensate for a single missing pair of alleles.nMost genes are pleiotropic.They are expressed in different tissues in different ways and at different times in development.Tissue-Specific Knockout MicenWhile housekeeping genes are expressed in all types of cells at all
23、stages of development,other genes are normally expressed in only certain types of cells when turned on by the appropriate signals(e.g.the arrival of a hormone).nTo study such genes,one might expect that the methods described above would work.However,it turns out that genes that are only expressed in
24、 certain adult tissues may nonetheless be vital during embryonic development.In such cases,the animals do not survive long enough for their knockout gene to be studied.The Cre/loxP SystemnOne of the bacteriophages that infects E.coli,called P1,produces an enzyme designated Cre that cuts its DNA into
25、 lengths suitable for packaging into fresh virus particles.Cre cuts the viral DNA wherever it encounters a pair of sequences designated loxP.All the DNA between the two loxP sites is removed and the remaining DNA ligated together again(so the enzyme is a recombinase).The Cre/loxP SystemUsing Method
26、1,mice can be made transgenic for nthe gene encoding Cre attached to a promoter that will be activated only when it is bound by the same transcription factors that turn on the other genes required for the unique function(s)of that type of cell;na target gene,the one whose function is to be studied,f
27、lanked by loxP sequences.The Cre/loxP SystemnIn the adult animal,nthose cells that nreceive signals(e.g.,the arrival of a hormone or cytokine)nto turn on production of the transcription factors needed nto activate the promoters of the genes whose products are needed by that particular kind of cellnw
28、ill also turn on transcription of the Cre gene.Its protein will then remove the target gene under study.nAll other cells will lack the transcription factors needed to bind to the Cre promoter(and/or any enhancers)so the target gene remains intact.nThe result:a mouse with a particular gene knocked ou
29、t in only certain cells.HIV-1 Proviral DNA Excision Using an Evolved Recombinase Science June 29,2007 nHIV-1 integrates into the host chromosome and persists as a provirus flanked by long terminal repeats(LTRs).To date,treatment regimens primarily target the virus enzymes or virus-cell fusion,but no
30、t the integrated provirus.We report here the substrate-linked protein evolution of a tailored recombinase that recognizes an asymmetric sequence within an HIV-1 LTR.This evolved recombinase efficiently excised integrated HIV proviral DNA from the genome of infected cells.Although a long way from use
31、 in the clinic,we speculate that this type of technology might be adapted in future antiretroviral therapies,among other possible uses.The use of transposon nIn gene trapnIn enhancer trapSchematic representation of the principle of trapping genes by the dual-tagging gene-trap method.n(A)A map of the
32、 pGT1 vector.The position and direction of Gal4,hs-neor,and mini-w genes are indicated by thick arrows.hspT represents the hsp70 terminator.3P and 5P are the P-element ends.The locations of the stop-start signal,splice donor site,and restriction sites are illustrated.(B)The structure of the gene-tra
33、p vector pGT1.A solid box represents the hsp70 terminator;an open circle,the splice acceptor site;and a solid circle,the splice donor site.Schematic representation of the principle of trapping genes by the dual-tagging gene-trap method.nWhen integrated into a gene in the Drosophila genome(C),pGT1 op
34、erates to produce two fusion transcripts,one encoding the GAL4 sequence fused to the 5 portion of the inserted gene,and the other encoding mini-w fused to the 3 portion of the gene(D).The fusion mRNA is translated into the functional proteins Gal4 and White(E),the former of which can activate transc
35、ription of a cDNA placed under the control of UAS(F),while the latter confers eye pigmentation in the fly signifying successful gene trapping.The mini-w genen The mini-w gene is followed by an artificial splice donor site,which is involved in the production of a chimeric mRNA composed of the mini-w
36、sequence and the exonic sequence of a host gene 3 to the vector integration site.This chimeric mRNA is polyadenylated and encodes the functional White protein.Even when the vector is inserted outside a gene,the mini-w gene can be transcribed from its own promoter.In this case,however,the mini-w mRNA
37、 would not be polyadenylated,because its 3 end cannot be spliced to an exon of the host gene.Such an unpolyadenylated mRNA is likely to be degraded rapidly before being translated,thereby resulting in the absence of eye pigmentation in the fly carrying the vector(the flies have a w-background).In co
38、ntrast,the mini-w gene is expected to confer a dark eye color on the fly when the vector is integrated into a gene,because the chimeric mRNA is polyadenylated.Efficient transposition of the piggyBac(PB)transposon in mammalian cells and mice.nCell.2005 Aug 12;122(3):473-83.nDing S,Wu X,Li G,Han M,Zhu
39、ang Y,Xu T.nInstitute of Developmental Biology and Molecular Medicine,School of Life Sciences,Fudan University,220 Handan Road,Shanghai 200433,China.a coinjection method to directly produce mice doubly positive for a nonautonomous transposon and a helper transposase gene.nTransgenic animals were pro
40、duced by conventional pronuclei injection of linear plasmids,which assured the cointegration of both donor and helper plasmids in the same locus.Several transgenic mouse lines carrying both PBAct-RFP and protamine 1(prm1)promoter-driven PB transposase transgenes(Prm1-PBase)were generated.The prm1 pr
41、omoter was expected to be active during spermiogenesis(OGorman et al.,1997).Thus,in such double-positive transgenic lines,male mice were expected to produce new transposition events whereas female mice could be used as breeders.Figure.Expression of Transgenes in piggyBac Vectorsn(A)PBAct-RFP express
42、ion in the progenies resulted in red fluorescence under the illumination of a portable long-wave UV light.Two positive mice(arrows)carrying the same single copy transposon(AF0-47T6)and two negative littermates(asterisks)are shown.n(B)PBAct-RFP expression in a founder mouse and her progeny.Red fluore
43、scence was mosaic in the founder.Segregation of transposons in the progeny resulted in different intensities of RFP signal.The star marks the transgene-negative littermate.Figure.Expression of Transgenes in piggyBac Vectors n(C and D)Coexpression of two transgenes in the same piggyBac vector.As a re
44、sult of tyrosinase expression,a PBK14-Tyr,Act-RFP founder shows gray coat color under white light,while the transgene-negative littermate remains albino(C,right and left,respectively).When illuminated by UV,red fluorescence was observed from this founder(D).nFUNCTIONAL GENOMICSnGain of functionnLoss
45、 of functionRNA InterferencenThe ability of dsRNA to suppress the expression of a gene corresponding to its own sequence is called RNA interference(RNAi).It is also called post-transcriptional gene silencing or PTGS.RNA InterferencenRNA interference(RNAi)is an evolutionarily conserved phenomenon in
46、which gene expression is suppressed by the introduction of homologous double-stranded RNAs(dsRNAs).After dsRNA molecules are delivered to the cytoplasm of a cell,they are cleaved by the RNase III-like enzyme,Dicer,to 21-to 23-nt small interfering RNAs(siRNAs).RNA InterferencenThese siRNAs are then i
47、ncorporated into a protein complex,the RNA-induced silence complex(RISC).The antisense strand of the duplex siRNA guides the RISC to the homologous mRNA,where the RISC-associated endoribonuclease cleaves the target mRNA,resulting in silencing of the target gene.nRNAi has been successfully used to su
48、ppress gene expression in a variety of organisms including zebrafish,Caenorhabditis elegans,Drosophila,planaria,mice,and mammalian cells.In C.elegans and Drosophila,RNAi is typically induced by the introduction of a long dsRNA(up to 12 kb)produced by in vitro transcription.This simple approach canno
49、t be used in mammalian cells,where introduction of long dsRNA elicits a strong antiviral response that obscures any gene-specific silencing effect.General Design Guidelines nIf you prefer to design your own siRNAs,you can choose siRNA target sites in a variety of different organisms based on the fol
50、lowing guidelines.Corresponding siRNAs can then be chemically synthesized,created by in vitro transcription,or expressed from a vector or PCR product.General Design Guidelinesn1.Find 21 nt sequences in the target mRNA that begin with an AA dinucleotide.nBeginning with the AUG start codon of your tra
