Plasmids
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Plasmids

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    Plasmids Plasmids Presentation Transcript

    • plasmids A plasmid is a circular, self-replicating DNA molecule carrying a few, useful but non necessary genes. Occurence procaryote organisms eukaryotic organisms like Entamoeba histolytica, yeast etc. Their size varies from 1 kbp to over 400 kilobase pairs (kbp). In a single cell there are anywhere from one copy, for large plasmids, to hundreds of copies of the same plasmid. We speaks of low and high copy number plasmids Plasmids are easy to manipulate and isolate from bacteria (kits). After being modified, they can be integrated into other genomes, plants, protists, mammals, thereby conferring to other organisms whatever genetic functionality they carry. Thus, this gives the ability to introduce genes into a given organism by using bacteria to amplify the hybrid genes that are created in vitro. This tiny but mighty plasmid molecule is the basis of recombinant DNA technology. ------------------------- to carry: llevar a few: un poco size: tamaño anywhere: en cualquier parte. whatever: cualquier -a
    • Bacterial plasmid
    • types Plasmids are classified 1. by their ability to be transferred to other bacteria Conjugative The sexual transfer of plasmids to another bacterium through a pilus. those plasmids possess the 25 genes required for transfert Non-conjugative Non-conjugative plasmids don’t initiate conjugaison. They can only be transferred with the help of conjugative plasmids. mobilisable An intermediate class of plasmids are mobilisable, and carry only a subset of the genes required for transfer. These plasmids can parasitise another plasmid, transferring at high frequency in the presence of a conjugative plasmid incompatibility groups: Several types of plasmids could coexist in a single cell. On the other hand, related plasmids are often incompatible, resulting in the loss of one of them from the cell line. ---------------------------- to classify: clasificar ability: aptitud, capacidad a subset is a set whose members are members of another set.
    • conjugative plasmids The sexual transfer of plasmids to another bacterium through a pilus. Those plasmids, plasmidos F, possess the 25 genes required for transfert. ---------------------------- El plásmido F contiene the 25 genes que controlan la producción de los pilis. Los pili se observan prácticamente solo en bacterias Gram negativas. transfert, m ¿ Cuál palabra escoger? in: They possess the genes required for transfert 1. (de prisionero, de población) traslado. 2. (de fondos, de mercancías) transferencia. 3. (de negocio) traspaso. 4. (de bienes inmuebles) transmisión. 5. Psicol transferencia
    • rolling circle replication:passes a plasmid through a pilus. A nice film showing how it works can be seen at:http://info.bio.cmu.edu/Courses/03441/TermPapers/2000TermPapers/group2/fig3.htmlWhat’s important in rolling circle replication is to find out the two 3’ ends used in the process
    • 2. by function1. Fertility-(F) plasmids, They are capable of conjugation (they contains the genes for the pili).2. Resistance-(R) plasmids, contain gene (s) that can build resistance against one or several antibiotics or poisons.3. Col-plasmids, contain genes coding for colicines, proteins that can kill other bacteria.4. Degradative plasmids, able to digest unusual substances, e.g., toluene or salicylic acid.5. Virulence plasmids, turn a bacterium into a pathogen.6. addiction system. These plasmids produce both a long-lived poison and a short-lived antidote. Daughter cells that retain a copy of the plasmid survive, while a daughter cell that fails to inherit the plasmid dies or suffers a reduced growth-rate because of the lingering poison from the parent cell.---------------------------------addiction: dependencia, adiccióndaughter: hijato inherit: heredar.lingering: persistente
    • Episomesare units of genetic material composed of a series of genes that sometimes has anindependent existence in a host cell and at other times is integrated into a chromosome of the cell,replicating itself along with the chromosome.Episomes exist in all organisms.In bacteria, episomes are: viruses:Bacteriophages in bacteria (lytic or lysogenic phages, prophages and proviruses) Sex factors Hfr:sex factors like the F factor F+ is autonomous, extrachromosomal.Hfr (or high frequency recombination) is the same sequence, but integrated into the host chromosome. Plasmids:can be integrated into a chromosome. transposons:Transposons are known as mobile genetic elements (they were discoverd in maize by Barbara McClintock*)While they can also exist outside of the chromosome, they prefer to, and are designed, to integrateinto the chromosome following their movement from one cell to another.For example, Class 1 transposons encode drug resistance genes.--------------------------host: huéspedhost cell, célula huésped
    • Integrons are mobile DNA elements able to capture genes, notably those encoding antibiotic resistance,by site-specific recombination.Class 1 integrons have been examined the most extensively. They consist of a variable regionbordered by 5 and 3 conserved regions. The 5 region is made up of the int1 gene, attI, and thepromoter Pr . Pr drives transciption of genes within the variable region. The 3 region consists ofqacED1*, coding a multiple substrates efflux pump, a sulfonamide resistance gene.The antibiotic resistance genes that integrons capture are located on gene cassettes. The cassettesconsist of a promoterless gene and a recombination site (attC). The cassettes can exist as free,circular DNA but cannot be replicated or transcribed in this form. A recombination event occurs betweenattI and attC, integrating the cassette into the integron. The gene on the cassette is then bound by theattI site on the 5 side and by attC on the 3 side.*http://www.biochemj.org/bj/376/0313/bj3760313.htm
    • conformation In bacteria, plamids are found in the supercoil (superenrollado) form. Supercoil s and relaxed (relajado) forms can be seen under the electron microscope, in electrophoresis or after centrifugation. The supercoiled form migrate and sediments quicky in in eletrophoresis and centrifugation respectively. Topoisomerases help to pass from one form to the other as we saw it earlier.
    • Plasmids in molecular biology Minimum requirements for plasmids useful for recombination technology: 1. Origin of replication (ORI). ORI enables a plasmid DNA to be duplicated independently from the chromosome 2. Selectable marker: allow to select for cells that have your plasmids. 3. Restriction enzyme sites in non-essential regions of the plasmid. Plasmid replication initiates in a cis-site called ori. It proceeds either by a rolling circle or a theta replication mechanism. Some of the plasmid-encoded elements required for their replication, such antisense RNA molecules and DNA repeated sequences located close to ori, determine plasmid attributes like copy number and incompatibility.
    • Plasmids often contain genes or gene-cassettes that confer a selective advantage when theyare inside a bacterium: resistance to antibiotics resistance to herbicides insecticide productionto the bacterium harboring them, for example, the ability to make the bacterium antibiotic resistant.
    • f1 origin The only difference between these two vectors is the orientaion of the f1 ori Either from right to left Or from left to right What is the f1 ori It is the origin of replication of the phage F1 Then, the vectors pGEM includes a bacteriophage f1 origin of replication: pGEM 3Zf1 (+) in one sens pGEM 3Zf1 (-) in the other sens.
    • Then, these vectors can be used for the production of single-stranded DNA (ssDNA) phages which were used for DNA sequencing. Each vector is used for the production of one strand.For induction of ssDNA, bacterial cells containing a pGEM-Zf recombinant areinfected with an appropriate helper phage. The plasmid enters the f1 replicationmode, and the resulting ssDNA is exported from the cells as an encapsidatedvirus particle. These particles were used for DNA sequencing.
    • pGEM-3Zf(+) and pGEM-3Zf(-) vectors share a common backbone and a multiplecloning site within lacZ sequences flanked by SP6 and T7 RNA polymerase promoters.These vectors differ only by which DNA strand is packaged with the additionof helper phage.For induction of ssDNA, bacterial cells containing a pGEM-Zf recombinant areinfected with an appropriate helper phage. The plasmid enters the f1 replicationmode, and the resulting ssDNA is exported from the cells as an encapsidatedvirus particle.----------------------------------Strand: hebra, cadena.backbone: la columna vertebralto flank: flanquear
    • Tranformation*In bacteria and yeast:transformation is the transfer of genetic information from a donor to a recipientusing naked DNA without cell to cell contact. The DNA from the donor is in themedium where it was secreted. The recipient takes this DNA up:there is no requirement for cell to cell contact.The only limitation of this uptake is the restiction system1. natural transformation (rare but dangerous): Many bacteria can acquire new genes by taking up DNA molecules (e.g., a plasmid) from their surrounding. While naturally transformable bacterial strains exist, these are sufficiently rare that induced transformation is more important to the geneticist. A captured DNA has to face restriction!2. experimental: in the lab, DNA is introduced by one of the following methods: a. Calcium chloride (followed by heat shock) method b. Electroporation. c. Liposomes----------------------------------------*in eucaryotes other than yeast, transformation is called transfection
    • In vitro transformation is made during rapid growth.DNA transformation is a process by which DNA can be transferred into yeast orbacterias. During rapid growth of E. Coli, a bacterium, the cell membrane hashundreds of pores, called adhesion zones. Transformation is made during thisRapid growth, i. e. when these pores are present.
    • calcium chorideUse E. coli after rapid growth*. 1. In a microtube, gently mix the plasmid DNA with the bacteria as you add it with a pipet. Incubate the mixture on ice for 20 minutes. 2. Heat shock the mixture by submitting it for 2 minutes at 42°C Transfer the bacterial suspension to 10 ml of L Broth without antibiotic. Shake for 45 minutes in a 37°C incubator. 3. Centrifuge the cells at 2200 rpm for 15-20 minutes at 4°C. Discard the supernatant. 4. Resuspend the bacteria in 100 µl of L broth (+ antibiotic) per Petri dish, and plate on a LB agar + antibiotic plates using the turntable and a sterile glass rod. 5. Determine the transformation efficiency the next day by counting the number of colonies-----------------------------------* It works better when bacterias are first treated with Calcium Chloride.
    • electroporation One way of physically introducing DNA into a cell is electroporation. The diagram shows an electrical circuit for a simple electroporation device. A solution of DNA fragments containing the gene of interest is added to the cuvette. The capacitor is charged by closing the right-hand switch. When the capacitor has been charged, the direct current pulse is discharged in the cuvette suspension by closing the left-hand switch. The DC pulse is thought both to disrupt temporarily the membrane and to “electrophorese” DNA into cells. ----------------------------- a switch: un interruptor capacitor : nm condensador reporter: aquí: un testigo. to close: cerrar pulse: impulse, descarga direct current (DC) : nf corriente continua
    • Electroporation SystemIntroduce DNA, RNA, and proteins into mammalian, plant, or bacterial cells.Most system come with the Pulse Control unit, Chamber Safe with a Chamber Rack,Electroporation Chambers, and a manual.Equipped with an internal power supply to deliver a range of reproducible electricalpulses to the cells by capacitor discharge.Several capacitors provide pulse lengths from <0.1 to >200 ms.Disposable electroporation chambers are available with a 0.4-cm inter-electrode gap(standard design) for transfecting mammalian or plant cells, or with a 0.15-cm inter-electrodegap (microelectroporation design) for transforming yeast or bacterial cells.
    • liposomes To physically introduce DNA into cells you can use liposomes. Liposomes are lipid-bilayer bounded vesicles. Produced by hydrating lipids in aqueous solutions. If DNA is present in the solution, it becomes incorporated into the liposomes. Liposomes interact with cell membranes. The liposomal contents is transferred to the inside of the cell. Both membrane fusion and endocytosis have been implicated as mechanisms. Genes present in the transferred DNA can be expressed transiently. The transferred DNA may also integrate into chromosomes and cell lines containing the integrated gene may be selected.
    • recombinant plasmids
    • T7 promoter Eco RI Hind III5’ tgtaatacgactcactatagggcgaattcgagctcggtacccggggatcctctagagtcgacctgcaggcatgcaagcttgagtattctatagttcacctaaat 3’3’ acattatgctgagtgatatcccgcttaagctcgagccatggg cccctaggagatctcagctggacgtccgtacgttcgaactcataagatatcaagtggattta 5’ SP6 promoterT7 and SP6 are (trans) RNA Pol from T7 and SP6 phages.They bound to to their cis promoters respectively and start making RNA. These RNA can ve used to make probes,used for micro-arrays or Southern blots.
    • (In between the T7 and SP6 promoter is the pUC 18 polylinker): At the left of the molecule is the ATG codon of the LacZ gene. The native nucleotide sequence of this plasmid is in small caps. A 54 nt fragment (large caps) interupts but doesn’t disrupt the reading frame 54 is a multiple of 3 !In the pGEM-3Z plasmid, in addition to the 54 nucléotide, Two promoters wereAlso added. T7 promoter Eco RI Hind III5’ tgtaatacgactcactatagggcgaattcgagctcggtacccggggatcctctagagtcgacctgcaggcatgcaagcttgagtattctatagttcacctaaat 3’3’ acattatgctgagtgatatcccgcttaagctcgagccatggg cccctaggagatctcagctggacgtccgtacgttcgaactcataagatatcaagtggattta 5’ SP6 promoter
    • opening of the plasmid polylinker: making room for the insertYou have to buy the plasmid, the restriction enzymes and their buffer. Eco RI Hind III5’ tgtaatacgactcactatagggcgaattcgagctcggtacccggggatcctctagagtcgacctgcaggcatgcaagcttgagtattctatagttcacctaaat 3’3’ acattatgctgagtgatatcccgcttaagctcgagccatggg cccctaggagatctcagctggacgtccgtacgttcgaactcataagatatcaagtggattta 5’ + Eco RI and Hind III 5’ aattcgagctcggtacccggggatcctctagagtcgacctgcaggcatgca 3’ 3’ gctcgagccatggg cccctaggagatctcagctggacgtccgtacgttcga 5’ 51 bp fragment to be eliminated, by electrophoresis for example +5’ tgtaatacgactcactatagggcg agcttgagtattctatagttcacctaaat 3’3’ acattatgctgagtgatatcccgcttaa actcataagatatcaagtggattta 5’ <-------------- place for an insert ------------>
    • The 51 bp fragment between Eco RI and Hind III is eliminated, by electrophoresisThe opened plasmid (2692 bp) is extracted from the gel either using agarase oran extraction kit. In each case, you should make the gel with LMP agarose.LMP means low melting point.
    • Trimming of the fragment we wanted to clone: Eco RI Hind III5‘ tatctggaattccctgccagtattatatgctgaattcagagattaagccaagcttgtgta 3‘3‘ atagaccttaagggacggtcataatatacgacttaagtctctaattcggttggaactctat 5‘ + Eco RI and Hind III5‘ aattccctgccagtattatatgctgaattcagagattaagcca 3‘3‘ gggacggtcataatatacgacttaagtctctaattcggttgga 5‘insertion of the fragment into the open plasmid 5‘aattccctgccagtattatatgctgaattcagagattaagcca 3‘ 3‘ gggacggtcataatatacgacttaagtctctaattcggttgga 5‘ +5’ tgtaatacgactcactatagggcg agcttgagtattctatagttcacctaaat 3’3’ acattatgctgagtgatatcccgcttaa actcataagatatcaagtggattta 5’5’ tgtaatacgactcactatagggcg aattccctgccagtattatatgctgaattcagagattaagcca agcttgagtattctatagttcacctaaat 3’3’ acattatgctgagtgatatcccgcttaa gggacggtcataatatacgacttaagtctctaattcggttgga actcataagatatcaagtggattta 5’+ Ligase and ATP
    • Ligation with ligase and ATP 5‘aattccctgccagtattatatgctgaattcagagattaagcca 3‘ 3‘ gggacggtcataatatacgacttaagtctctaattcggttgga 5‘ +5’ tgtaatacgactcactatagggcg agcttgagtattctatagttcacctaaat 3’3’ acattatgctgagtgatatcccgcttaa actcataagatatcaagtggattta 5’5’ tgtaatacgactcactatagggcg aattccctgccagtattatatgctgaattcagagattaagcca agcttgagtattctatagttcacctaaat 3’3’ acattatgctgagtgatatcccgcttaa gggacggtcataatatacgacttaagtctctaattcggttgga actcataagatatcaagtggattta 5’After ligation, the plasmid and the fragment it contains are a “recombinant DNA”5’ tgtaatacgactcactatagggcgaattccctgccagtattatatgctgaattcagagattaagccaagcttgagtattctatagttcacctaaat 3’3’ acattatgctgagtgatatcccgcttaagggacggtcataatatacgacttaagtctctaattcggttggaactcataagatatcaagtggattta 5’
    • lactose operon This plasmid do alpha complementation, it allows what people call white and blue screening. This plamid is a bit strange, why in the world would we insert the DNA fragment in the midle of something called lacZ ? Inserting something over there would disturb the lacZ sequence and inactivate it. lacZ code for a protein, the alpha peptide which is necessary for ß-galactosidase to work.. This plasmid is used to transform cells lacking the lacZ sequence. When the plasmide is inside such a bacterium it will “complement” it with this lacZ sequence. The plasmid affords the sequenceto the bacterium: it complement it
    • The gene Z code for ß-galactosidase.The sequence of this gene can be cut in two parts.The proteins coded by the two parts (alpha and beta) should be present for ß-galactosidaseactivity. The sum of the two polypeptides have the same activiy as the whole gene product.Some mutated bacteria lack the alpha part: they don’t show any ß-galactosidase activity.When these bacterias are grown with X-gal, their colony stay white.When they are transformed by a plasmid containing the lac Z gene (coding the alpha part).The plasmid afford the missing part; the clonies become blue when x-gal is in the culturemedium.If a DNA fragment is inserted in the plasmid, it interrupts lacZ, and the colonies stay white.
    • A cell without plasmid would die in the presence of ampicillin, because it needa gene to detroy the ampicillin in the medium.The plasmid provide this gene.
    • If the plamid has no insert, the bacterium would produce an efficient ß-galactosidase,And so, be able to hydrolyse X-Gal: the colony will be blueIf the plasmid has an insert, the alpha peptide will be absent, and, in presence of X-gal,the colonies would remain white.The the white colonies have a plamid with an insert.
    • mammalian plasmids Some plasmids are modified so that they can replicate or transcribe in other hosts: Bacterial plasmids can be engineered to be transfected in mammalian cells. In bacterias, genes are polycistronic. In mammalian cells, genes are mostly monocistronic. The promoter should be a good mammalian promoter ( the promoter of a mammalian viruses, like Cytomegalo virus or SV40 virus) . An intron is also added. To construct the poly A tail of the mRNA a polyadenylation signal is added. The cDNA to be expressed is inserted between the intron and the polyadenylation signal.
    • Transduction is the process by which genetic material, e.g. DNA or siRNA, is inserted into a cell. Common techniques in molecular biology are the use of viral vectors (including bacteriophages), electroporation, or chemical reagents that increase cell permeability. Transfection and transformation are more common terms, although these sometimes imply expression of the genetic material as well. Akt = PKB Akt-P = Ser-473-phosphorylated Ak p27: This gene encodes a cyclin-dependent kinase inhibitorFigure 2. Expression levels of various proteins following PTEN transductioninto glioblastoma cell line A172. PTEN is highly expressed after transduction.Total Akt protein expression has not changed following PTEN transduction,whereas levels of actived Akt (Ser-473-phosphorylated Akt) are reduced afterPTEN transduction. Conversely, p27 protein accumulates after PTEN transduction.`ß-actin expression remains constant, showing that equal amounts of protein wereloaded in each wellINTERNATIONAL JOURNAL OF ONCOLOGY 21: 1141-1150, 2002Genetic profile, PTEN mutation and therapeutic role of PTEN in glioblastomas XING FAN,YAN AALTO, STEPHEN G. SANKO, SAKARI KNUUTILA, DAVID KLATZMANN andJAVIER S.CASTRESANA
    • GFPThe phMGFP Vector contains the open reading frame for the Monster Green Fluorescent Proteincloned into a mammalian expression vector. The MGFP is encoded by an improved synthetic versionof thegreen fluorescent protein gene originally cloned from Montastrea cavernosa(Great Star Coral).CMV Enhancer/Promoter: This CMV enhancer/promoter allows strong, constitutive expression.Chimeric Intron: is composed of the5´-donor site from the first intron of the human β-globin and the branch and3´- acceptor site from the intron between the leader and body of an immunoglobin geneheavy chain variable region. Transfection studies have demonstrated that the presence ofan intron flanking the cDNA insert frequently increases the level of gene expressionT7 Promoter: A T7 RNA polymerase promoter is located downstream of the chimeric intron
    • Green fluorescent protein (GFP) is commonly used to monitor gene expression andprotein trafficking within intact cells. GFP fusion proteins are easily visualized bystandard fluorescence microscopy to track realtime subcellular localization of aprotein of interest.
    • Luc The pGL2 Luciferase Reporter Vectors(a) provide a basis for the quantitative analysis of factors that potentially regulate mammalian gene expression. These factors may be cis-acting, such as promoters and enhancers, or trans-acting, such as various DNA-binding factors. The pGL2 Vectors carry the coding region for firefly ( Photinus pyralis) luciferase, used to monitor transcriptional activity in transfected eukaryotic cells.
    • CAT The pCAT®3-Basic Vector lacks eukaryotic promoter and enhancer sequences, allowing maximum flexibility in cloning putative regulatory sequences. Expression of CAT activity in cells transfected with this plasmid depends on insertion and proper orientation of a functional promoter upstream from the intron and the CAT gene. Potential enhancer elements can also be inserted upstream of the promoter or in the BamH I or Sal I sites downstream of the CAT transcription unit.
    • trypanosomal plasmids
    • plasmids of E. histolytica
    • plant plasmids and GMO Large gall formed at the base of the stem of a rose bush Agrobacterium causes Crown gall disease. This disease occurs when the soil bacterium Agrobacterium tumefaciens (A. tumefaciens ) enters a stem through a wound site (crown is the stem just above the soil surface). This causes proliferation of tissue, like cancer growth (gall) and can occur on many dicot species (grape, fruit and nut trees etc.). Basically, the bacterium transfers part of its DNA to the plant, and this DNA integrates into the plant’s genome, causing the production of tumours and associated changes in plant metabolism. http://helios.bto.ed.ac.uk/bto/microbes/crown.htm
    • The unique mode of action of A. tumefaciens has enabled this bacterium to be used asa tool in plant breeding. Any desired genes, such as insecticidal toxin genes or herbicide-resistance genes,can be engineered into a bacterial plasmid, the Ti plasmid and thereby inserted into the plant genome.Most of the genes involved in crown gall disease are not borne on the chromosome of A. tumefaciensbut on a large plasmid, termed the Ti (tumour-inducing) plasmid. In the same way, most of the genesthat enable Rhizobium strains to produce nitrogen-fixing nodules are contained on a large plasmidtermed the Sym (symbiotic) plasmid. Thus, the characteristic biology of these two bacteria is afunction mainly of their plasmids, not of the bacterial chromosome. In natural conditions, the motile cells of A. tumefaciens are attracted to wound sites by chemotaxis(acetosyringone 10-7 Molar). Acetosyringone plays a further role in the infection process, because at higherconcentrations (about 10-5 to 10-4 Molar) than those that cause chemotaxis it activates the virulencegenes (Vir genes) on the Ti plasmid. These Vir genes coordinate the infection process; they: 1. lead to the production of proteins (permeases) that are inserted in the bacterial cell membrane for uptake of nutritive compounds (opines) that will be produced by the tumours 2. cause the production of an endonuclease - a restriction enzyme - that excises part of the Ti plasmid termed the T-DNA (transferred DNA).
    • When integrated into the plant genome, the genes on the T-DNA code for: * production of cytokinins * production of indoleacetic acid * synthesis and release of novel plant metabolites - the opines and agrocinopines.The plant hormones upset the normal balance of cell growth, leading to the production of gallsand thus to a nutrient-rich environment for the bacteria. The opines are unique amino acid derivatives,different from normal plant products, and the agrocinopines similarly are unique phosphorylatedsugar derivatives. All these compounds can be used by the bacterium as the sole carbon and energy source,and because they are absent from normal plants they provide Agrobacterium with a unique food sourcethat other bacteria cannot use.
    • Genetic engineering of plants with A. tumefacien
    • ess the gene for polygalacturonase, an enzyme that degrades pectin,s. As a result, the tomatoes can be left on the plant for longernd they also give a better consistency of tomato pastes.eered to express the insecticidal toxin gene of Bacillus thuringiensis , so that insects attempting to eat these plants are killed. potential disadvantage that continuous exposure of insects toent of toxin resistance. Cropping strategies must be devised ered for resistance to herbicides such as glyphosate, so that the herbicide can be used for weed control without damaging theplored or commercialised include: corporation of viral coat protein genes or antisense RNApathogens, by enhanced expression of fungal wall-degrading enzymes (chitinase and glucanases) a late stage in the development of their seeds, they express a gene that renders the seeds sterile. This "terminator technology"
    • gene knockout A gene knockout is a genetically engineered organism that carries one or more genes in its chromosomes that has been made inoperative. So far such organisms have been engineered chiefly for research purposes. Knockout is accomplished through a combination of techniques, beginning in the test tube with a plasmid, a bacterial artificial chromosome or other DNA construct, and proceeding to cell culture Individual cells are genetically transformed with the construct and--for knockouts in multi-cellular organisms--ultimately fused with a stem cell from a nascent embryo. The construct is engineered to recombine with the target gene, which is accomplished by incorporating sequences from the gene itself into the construct. Recombination then occurs in the region of that sequence within the gene, resulting in the insertion of a foreign sequence to disrupt the gene. With its sequence interrupted, the altered gene in most cases will be translated into a nonfunctional protein, if it is translated at all.
    • homologous recombination: Suppose that we wanted to replacethe sequence in red by nother one. 1 tatctggttg atcctgccag tattatatgc tgatgttaga gattaagcca tgcatgtgta 61 agtataaaga ccaagtagga tgaaactgcg gacggctcat tataacagta atagtttctt 121 tggttagtaa agtacaagga tagctttgtg aatgataaag ataatacttg agacgatcca 181 atttgtatta gtacaaagtg gccaatttat gtaagtaaat tgagaaatga cattctaagt 241 gagttaggat gccacgacaa ttgtagaaca cacagtgttt aacaagtaac caatgagaat 301 ttctgatcta tcaatcagtt ggtagtatcg aggactacca agattataac ggataacgag 361 gaattggggt tcgacatcgg agagggagct ttacagatgg ctaccacttc taaggaaggc 421 agcaggcgcg taaattaccc actttcgaat tgaagaggta gtgacgacac ataactctag 481 agttgagtaa aatcaattct tgaaggaatg agtaggaggt aaattctcct acgaaatcaa 541 ttggagggca agtctggtgc cagcagccgc ggtaattcca gctccaatag tgtatattaa 601 agttgctgtg attaaaacgc tcgtagttga attaaaatgt gattttatac attttgaaga 661 ctttanntaa gtgaagtttc tagaaatgtt aaattaaaat caaagaagga gacnnttcaa 721 gtaattgagt tgttattact ttgaataaaa taaggtgttt aaagcaaaac attatgttaa 781 tgaatattcg agcatgggac aatgctgagg ggatgtcaat tagacatttc gagagaagga 841 ttaaaaggaa caattggggt gattcagaaa ataacgggag aggtgaaaat ccatgatcgg 901 tataagatgc acgagagcga aagcatttca ctcaactggg tccattaatc aagaacgaaa 961 gttaggggat cgaagacgat cagataccgt cgtagtccta actataaacg atgtcaacca 1021 aggattggat gaaattcaga tgtacaaaga tgaagaaaca ttgtttctaa atccaagtat 1081 atcaatacta ccttgttcag aacttaaaga gaaatcttga gtttatggac ttcaggggga 1141 gtatggtcac aaggctgaaa cttaaaggaa ttgacggaag ggcacaccag gagtggagcc 1201 tgcggcttaa tttgactcaa cacgggaaaa cttaccaaga ccgaacagta gaaggaatga 1261 cagattaaga gttctttcat gatttattgg gtagtggtgc atggccgttc ttagttggtg 1321 gagtgatttg tcaggttaat tccggtaacg aacgagactg aaacctatta attagttttc 1381 tgcctataag acagaaatgt tcgcaagaac aggtgcgtaa gtaccacttc ttaaagggac 1441 acatttcaat tgtcctattt taattgttag ttatctaatt tcgattagaa ctcttttaac 1501 gtgggaaaaa gaaaaaggaa gcattcagca ataacaggtc tgtgatgccc ttagacatct 1561 tgggccgcac gcgcgctaca atggagttac tagagagcat tttatcattt acaccttatt 1621 tattaggcta tgtctaatag gtagggatag taagtggtgt accgagattg aaatagttaa 1681 ggaaaactca aaagaacgta catgacaggg ataaatgatt ggaattattt gttttgaacg 1741 aggaattcct tgtaatatcg agtcattaac tcgagatgaa tacgtccctg ccctttgtac 1801 acaccgcccg tcgctcctac cgattgaata aagaggtgaa attctaggat tctgtcttat 1861 agatagaaaa atggatttaa atctccttat ttagaggaag gagaagtcgt aacaaggttt 1921 ccgtaggtga acctgcggaa ggatcatta
    • Regardless of what you want to insert, you must include some flanking DNA that is identicalin sequence to the targeted locus. Lets color them in blue and green respectively. Let’s representsequence in the next drawings by a blue, a red and a green line. 1 tatctggttg atcctgccag tattatatgc tgatgttaga gattaagcca tgcatgtgta 61 agtataaaga ccaagtagga tgaaactgcg gacggctcat tataacagta atagtttctt 121 tggttagtaa agtacaagga tagctttgtg aatgataaag ataatacttg agacgatcca 181 atttgtatta gtacaaagtg gccaatttat gtaagtaaat tgagaaatga cattctaagt 241 gagttaggat gccacgacaa ttgtagaaca cacagtgttt aacaagtaac caatgagaat 301 ttctgatcta tcaatcagtt ggtagtatcg aggactacca agattataac ggataacgag 361 gaattggggt tcgacatcgg agagggagct ttacagatgg ctaccacttc taaggaaggc 421 agcaggcgcg taaattaccc actttcgaat tgaagaggta gtgacgacac ataactctag 481 agttgagtaa aatcaattct tgaaggaatg agtaggaggt aaattctcct acgaaatcaa 541 ttggagggca agtctggtgc cagcagccgc ggtaattcca gctccaatag tgtatattaa 601 agttgctgtg attaaaacgc tcgtagttga attaaaatgt gattttatac attttgaaga 661 ctttanntaa gtgaagtttc tagaaatgtt aaattaaaat caaagaagga gacnnttcaa 721 gtaattgagt tgttattact ttgaataaaa taaggtgttt aaagcaaaac attatgttaa 781 tgaatattcg agcatgggac aatgctgagg ggatgtcaat tagacatttc gagagaagga 841 ttaaaaggaa caattggggt gattcagaaa ataacgggag aggtgaaaat ccatgatcgg 901 tataagatgc acgagagcga aagcatttca ctcaactggg tccattaatc aagaacgaaa 961 gttaggggat cgaagacgat cagataccgt cgtagtccta actataaacg atgtcaacca 1021 aggattggat gaaattcaga tgtacaaaga tgaagaaaca ttgtttctaa atccaagtat 1081 atcaatacta ccttgttcag aacttaaaga gaaatcttga gtttatggac ttcaggggga 1141 gtatggtcac aaggctgaaa cttaaaggaa ttgacggaag ggcacaccag gagtggagcc 1201 tgcggcttaa tttgactcaa cacgggaaaa cttaccaaga ccgaacagta gaaggaatga 1261 cagattaaga gttctttcat gatttattgg gtagtggtgc atggccgttc ttagttggtg 1321 gagtgatttg tcaggttaat tccggtaacg aacgagactg aaacctatta attagttttc 1381 tgcctataag acagaaatgt tcgcaagaac aggtgcgtaa gtaccacttc ttaaagggac 1441 acatttcaat tgtcctattt taattgttag ttatctaatt tcgattagaa ctcttttaac 1501 gtgggaaaaa gaaaaaggaa gcattcagca ataacaggtc tgtgatgccc ttagacatct 1561 tgggccgcac gcgcgctaca atggagttac tagagagcat tttatcattt acaccttatt 1621 tattaggcta tgtctaatag gtagggatag taagtggtgt accgagattg aaatagttaa 1681 ggaaaactca aaagaacgta catgacaggg ataaatgatt ggaattattt gttttgaacg 1741 aggaattcct tgtaatatcg agtcattaac tcgagatgaa tacgtccctg ccctttgtac 1801 acaccgcccg tcgctcctac cgattgaata aagaggtgaa attctaggat tctgtcttat 1861 agatagaaaa atggatttaa atctccttat ttagaggaag gagaagtcgt aacaaggttt 1921 ccgtaggtga acctgcggaa ggatcatta
    • The next step is to design and fabricate the DNA construct you want to insert in place of the red sequence.This construct may contain any DNA sequence, but must include some flanking DNA (blue and green).In addition to the positive selection marker (e.g. antibiotic resistance) often a negative selection marker(e.g. thymidine kinase, tk) is added to the replacement vector.The negative marker is outside the region of sequence similarity between the vector and the targeted locus. The positive selection marker, neo, code for a protein that inactivates G418, a neomycine homolog. The negative marker code for thymidine kinase (tk). If tk is incorpotated, this is due to a non-homologous recombination event. In this case, tk kill the cell by modifying the molecules of gancyclovir added to the medium So the cell in which happen the right recomnination will stay alive in a medium containing Neomycin and gancyclovir.
    • During meiosis and mitosis when homolgous chromosomes align along the metaphase plane,the engineered construct finds the targeted gene and recombination takes place within the homolgous(meaning identical in this case) sequences The recombination may take place anywhere within theflanking DNA sequences and the exact location is determined by the cells and not the investigators.Once the cells have performed their part of the procedure,the end result is a new piece of DNA inserted into the chromosome. The rest of the genome is unalteredbut the single targeted locus has been replaced with the engineered construct and some of its flankingDNA.The original engineered construct has taken up the targeted gene of interest but since it cannot replicatein a nucleus, it is lost quickly in dividing cells while the modified chromosome replicates faithfully,including its new insert.Cells that have undergone homologous recombination can be selected by addition of antibioticto the growth medium (positive selection). Notice that the negative selection marker is not incorporatedinto the chromosome by homologous recombination.
    • Look now at the green sequences which were addded on the drawing. This sequence recognizethe long and the short fragments:Bam HI -Bam HI 5.5 kb and Bam HI -Bam HI 15 kbWe not only synthetize this fragment, but in addition we labelled it with 32P or 33P, both areradioactive isotopes of phosphorous (It’s time for you to study how we do that!)Then, we prepare and purify the DNA of this organism. If the organism is a mouth, weprepare genomic DNA from a tail fragment.We hydrolyse completely this DNA with Bam HI to be sure to get the two fragments:Bam HI -Bam HI 5.5 kb and Bam HI -Bam HI 15 kbWe submit the hydrolysate to the Southern Analysis.
    • after an electrophoresis on agarose gel and denaturation (separation of the two strands),the gel is put on a device which allow the tranfer of the DNA on a nylon filter. The denaturedDNA is attached covalently to the membrane by a short UV irradiation(both strand are stillseparated).The filter is put in a freezer bag. The (green) labelled probe is added.The probe binds to its complement either the 15 or the 4.5 fragment