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Chapter 17 Gene Technology Central Dogma: DNA -> RNA -> Protein DNA CCTGAGCCAACTATTGATGAA transcription RNA CCUGAGCCAACUAUUGAUGAA translation Protein PEPTIDE Genetic Recombination in Humans There are three ways in which meiosis and fertilization ensure that a child has a different combination of genes from that of either parent: 1. Independent assortment of chromosomes during metaphase I 2. Crossing-over during prophase I 3. Upon fertilization, recombination of chromosomes from different individuals (via their gametes) occurs. Recombinant DNA technology or genetic engineering was developed in 1971-1973 Their core was gene cloning Lead to DNA sequencing techniques that enabled the structures of individual genes to be determined Lead to procedures for studying the regulation of individual genes Genetic recombination - transfer of DNA from one organism (donor) to another recipient. The transferred donor DNA may then be integrated into the recipient's nucleoid by various mechanisms (homologous, non-homologous). Types Of Recombination Generalized or Homologous Recombination Occurs during prophase of meiosis I and involves exchange between homologous strands of DNA Site Specific Recombination - Short homologous sections of bacterial and phage DNA serve as a site for recombination and thus incorporation of phage DNA into bacterial chromosomes Transposition - Not truly recombination between different genomes, but the movement of transposons within a genome Homologous recombination homologous DNA sequences having nearly the same nucleotide sequences are exchanged by means of Rec A proteins. This involves breakage and reunion of paired DNA segments as seen in Natural mechanisms of genetic recombination in bacteria include: a. transformation b. transduction c. conjungation The Current Prokaryotic Recombination Model 5´ 3´ 3´ 5´ 3´ 5´ 5´ 5´ 3´ 3´ 5´ 3´ 5´ 5´ 3´ 5´ 3´ 3´ 3´ 3´ 内切酶 (recBCD) 3´ 5´ 5´ 3´ 内切酶 (recBCD) 5´ 3´ 3´ 5´ 3´ 5´ 5´ 3´ 5´ 3´ DNA侵扰 (recA) 分支迁移 (recA) DNA 连接酶 5´ 3´ 3´ 5´ 3´ 5´ 5´ 3´ 5´ 3´ 3´ 5´ 3´ 5´ 5´ 3´ Holiday中间体 目录 5´ 3´ 3´ 5´ 3´ 5´ 5´ 3´ Holiday中间体 3´ 5´ 5´ 3´ 3´ 5´ 5´ 3´ 内切酶 (ruvC) 3´ 3´ 3´ 5´ 拼 5´ 3´ 5´ 5´ 3´ 3´ 5´ DNA 连接酶 5´3´ 3´ 5´ 5´ 3´ 5´ 5´ 3´ 5´ 3´ 内切酶 (ruvC) 接 重 组 体 体片 段 重 组 splice recombination 5´ 3´ DNA 连接酶 3´ 5´ 3´ 5´ 3´ 3´ 5´ Patch recombination 5´ Genetic Transfer & Recombination In Bacteria Three kinds of genetic exchanges between prokaryotes Three kinds Conjugation Mediated by plasmids Transformation Mediated by free DNA Transduction Mediated by phages Bacterial Conjugation Bacterial Conjugation is genetic recombination in which there is a transfer of DNA from a living donor bacterium to a recipient bacterium. Often involves a sex pilus. The 3 conjugative processes + I. F conjugation II. Hfr conjugation III. Resistance plasmid conjugation I. F+ Conjugation Process F+ Conjugation- Genetic recombination in which there is a transfer of an F+ plasmid (coding only for a sex pilus) but not chromosomal DNA from a male donor bacterium to a female recipient bacterium. Involves a sex (conjugation) pilus. Other plasmids present in the cytoplasm of the bacterium, such as those coding for antibiotic resistance, may also be transferred during this process. Conjugal transfer of plasmid The 4 stepped F+ Conjugation 1. The F+ male has an F+ plasmid coding for a sex pilus and can serve as a genetic donor 2. The sex pilus adheres to an Ffemale (recipient). One strand of the F+ plasmid breaks The 4 stepped F+ Conjugation (cont’d) 3. The sex pilus retracts and a bridge is created between the two bacteria. One strand of the F+ plasmid enters the recipient bacterium F 4. Both bacteria make a complementary strand of the F+ plasmid and both are now F+ males capable of producing a sex pilus. There was no transfer of donor chromosomal DNA although other plasmids the donor bacterium carries may also be transferred during F+ conjugation. http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/conjugation/f.htm l Transformation Genetic recombination in which a DNA fragment from a dead, degraded bacterium enters a competent recipient bacterium and it is exchanged for a piece of the recipient's DNA. Involves 4 steps http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/transformation/transformation.html The 4 steps in Transformation 1. A donor bacterium dies and is degraded 2. A fragment of DNA from the dead donor bacterium binds to DNA binding proteins on the cell wall of a competent, living recipient bacterium 3. The Rec A protein promotes genetic exchange between a fragment of the donor's DNA and the recipient's DNA 4. Exchange is complete Transduction Genetic recombination in which a DNA fragment is transferred from one bacterium to another by a bacteriophage Structure of T4 bacteriophage Contraction of the tail sheath of T4 What are Bacteriophages? Bacteriophage (phage) are obligate intracellular parasites that multiply inside bacteria by making use of some or all of the host biosynthetic machinery (i.e., viruses that infect bacteria An infection cycle Transduction There are two types of transduction: generalized transduction: A DNA fragment is transferred from one bacterium to another by a lytic bacteriophage that is now carrying donor bacterial DNA due to an error in maturation during the lytic life cycle. specialized transduction: A DNA fragment is transferred from one bacterium to another by a temperate bacteriophage that is now carrying donor bacterial DNA due to an error in spontaneous induction during the lysogenic life cycle Site Specific Recombination Short homologous sections of bacterial and phage DNA serve as a site for recombination and thus incorporation of phage DNA into bacterial chromosomes att = attachment site O = center core of 15 bases = the same in phage & bacterial dsDNA B,P = different in size and sequence in bacterial & phage XIS = Excisionase INT = integrase Integration of Lambda DNA-overview. The control of INT & XIS activity determines it latency or not. Integration of Lambda DNA-Detail of crossover 例:细菌的特异位点重组 沙门氏菌H片段倒位决定鞭毛相转变 Transposition -Movement of gene to a new site, on same or a different chromosome Does not require extensive homology Transposable elements Insertion sequence 插 入 序 列 的 复 制 性 转 座 目录 Transposons: Mobile genetic elements that enable genes to move between non-homologous sites in DNA – Transposable elements. ⋅Altered expression of genes in new environments Gene technology A set of methods and techniques used to study biological processes on the molecular level There have been considerable developments in this field during the past two decades Eg: new and powerful ways for the isolation, analysis, and manipulation of nucleic acids What is gene cloning The basic steps in gene cloning experiment are as follows: (1) A fragment of DNA is inserted into a circular DNA molecule called a vector, to produce a chimera or recombinant DNA molecule (2) The vector acts as a vehicle that transports the gene into a host cell (3) Within the host cell the vector multiplies, producing numerous identical copies (4) When the host cell divides, copies of the recombinant DNA molecule are passed to progeny and further vector replication takes place (5) After a large number of cell divisions, a colony, or clone, of identical host cells is produced Each cell in the clone contains one or more copies of the recombinant DNA molecule The gene carried by the recombinant molecule is now said to be cloned The basic steps in gene cloning Why gene cloning is so important This technique can provide a pure sample of individual gene, separated from all the other genes in the cell Gene isolation by cloning Cloning allows individual fragments of DNA to be purified isolated long genes or those that have never been studied before Cloning allows individual fragments of DNA to be purified The basic concepts about gene cloning Clone A large population of identical molecules, or cells that arise from a common ancestor. Cloning The process that produces a large number of DNA or cell copies vector target DNA Recombination DNA Transformation bacteria Transfer the bacteria to a solid culture plate Screening the bacteria containing the recombinant DNA The process of gene cloning with the plasmid as vector DNA recombination The process that two DNA molecules from different source join together by covalent bond to form a new DNA molecule is called DNA recombination. Recombinant DNA DNA recombination technique By the application of some tool enzymes, the target gene and vector are ligated together, then introduced into the recipient cells which multiply and express the protein products coded by the target gene, that is, DNA recombination technique, DNA cloning or Gene cloning, Molecular cloning. Genetic engineering All the work or methods used related to the gene cloning and the target gene expressed in host cells to produce the special protein or polypeptide, or to change the character of an organism, are called genetic engineering. The requirements for the gene cloning (1) The target genes (2) The vectors (3) The tool enzymes (4) The host cells Target DNA • cDNA The cDNA are synthesized by reverse transcriptionase based on their mRNA templates of a cell line or tissue. • Genomic DNA It represents whole DNA sequence of a genome Purification of DNA from living cells Preparation of total cell DNA (RNA) Preparation of plasmid DNA Preparation of bacteriophage DNA Vectors Cloning vector——Cloning vectors are DNAs which can carry target genes, transfer them into the recipient cells. Cloning vector classes Plasmid DNA Phage DNA Virus DNA As for the expression vectors, they can make the proteins which are coded by the target gene expressed in the host cell Plasmids Basic features of plasmids Small (less than 10kb), Circular, duplex molecules of DNA Exist at low or high copies within the bacteria, but useful plasmid present in multiple copies Replicate independently from the bacterial cell Contain selectable markers, eg: the antibiotic resistance capability conferred to bacterium Possess at least one DNA sequence that act as an origin of replication Multiple RE sites ( multiple cloning sites, MCS) plasmid (质粒 ) b Go to pBR322 Fig pBR322 Multiple cloning site (MCS) Lac Z β-galactosidase gene Ampicillin resistance gene Origin of replication Fig pUC19 Go to pUC Bacteriophages Basic features of bacteriophages Bacteriophages, or phages are viruses that specifically infect bacteria Simple in structure, merely of a DNA (or occasionally RNA) carrying genes, including several for replication of the phage, surrounded by a protective coat or capsid made up of protein The general pattern of infection Attaches to the outside of the bacterium and injects its DNA chromosome into the cell The phage DNA is replicated, usually by specific phage enzymes coded by genes on the phage chromosome Other phage genes direct synthesis of the protein components of the capsid, new phage particles are assembled and released The result of Infection Lytic cycle: With some phage types the entire infection cycle is completed very quickly, possibly in less than 20 min. This type of rapid infection is called lytic cycle. Lysogenic infection: Characterized by retention of the phage DNA molecule in the host bacterium, possibly for many thousands of cell divisions Common used phages Bacteriophage λ A linear dsDNA approximately 49 Kb in length After infection it forms circular structures The phage DNA is inserted into the bacterial genome The first two classes of vector to be produced were λ insertion (λgt phages) phages) and λ replacement (EMBL Bacteriophage M13 A circular ssDNA, and has been used for sequencing of a cloned target DNA fragment Multiple cloning site (MCS) Lac Z β-galactosidase gene Ampicillin resistance gene Origin of replication Fig pUC19 Go to pUC Other vectors Cosmid (粘性质粒) Other vectors Bacterial artificial chromosome (BAC) and yeast chromosome Viruse are used as vectors, eg: retro-virus, adeno-virus, adenoassociated virus, etc The tool enzymes Nucleases—cut, shorten or degrade nucleic acid molecules Ligases—join nucleic acid molecules together Polymerase—make copies of molecules Modifying enzymes —remove or add chemical groups Topoisomerases —introduce or remove supercoils from covalently closed-circular DNA Nucleases degrade DNA molecules by breaking the phosphodiester bonds There are two different kinds of nucleases Exonucleases remove nucleotides one at a time from the end of a DNA molecule Endonucleases are able to break internal phosphodiester bonds within a DNA molecule ligases To repair single-stranded breaks(discontinuities) that arise in double-stranded DNA molecules during DNA replication Join together two individual fragments of double-stranded DNA Polymerases Synthesize a new strand of DNA complementary to an existing DNA or RNA template Four types of DNA polymerase are used routinely in genetic engineering DNA polymerase I: from E.coli. Synthesizes dsDNA by formation of a 5’,3’-phosphodiester bond Klenow fragment : removes the first 323 amino acids from DNA polymerase I , Synthesizes DNA by formation of a 5’,3’-phosphodiester bond Reverse transcriptase: synthesizes DNA from RNA template Taq DNA polymerase: used in the PCR, it is the DNA polymerase I from bacterium Thermus aquaticus DNA modifying enzymes Alkaline phosphatase from E.coli, calf intestinal tissue or arctic shrimp removes the phosphate group present at the 5’terminus of a DNA molecule Polynucleotide kinase from E.coli infected with T4 phage Has the reverse effect of alkaline phosphatase, adding phosphate groups onto free 5’termini DNA modifying enzymes Terminal deoxynucleotidyl transferase from calf thymus tissue adds one or more deoxyribonucleotides onto the 3’ terminus of a DNA Topoisomerases Change the conformation of covalently closedcircular DNA by introducing or removing supercoils. Enzymes for cutting DNA-restriction endonucleases The initial observation that led to the eventual discovery of restriction endonucleases (RE) was made in the early 1950s Restriction occurs because the bacterium produces an enzyme (called restriction endonucleases) that degrades the phage DNA Three different classes of RE are recognized, but the most important one is RE II which is used in DNA manipulation The discovery of these enzymes led to Nobel prizes for W.Arber, H. Smith and D. Nathans in 1978 Type II restriction endonucleases (RE) cut DNA at specific nucleotide sequences Generally, 4~8 bases be found, mostly 6 bases, a few of 8~10 bases The sequences discriminated usually are palindrome structure To cut the double strands of DNA at special sites and to yield two kinds of ends: blunt ends and sticky ends Blunt ends and sticky ends: Sticky or cohesive ends: the cleavage is staggered by two or four nucleotides the resulting DNA fragments have short single- stranded overhangs at each end Base pairing between them can stick the DNA molecule back together again Restriction endonucleases with different recognition sequences may produce the same sticky ends eg: BamH I (GGATCC) and Bgl II (AGATCT) 5’-sticky end (EcoR I ) 5’-GGTGAATTCAGC…-3’ 3’-CCACTTAAGTCG…5’ 5’-GGTG 3’-CCACTTAA + AATTCAGC…-3’ GTCG…5’ 3’-sticky end ( Pst I ) 5’-TTGCTGCAGAAG…-3’ 3’-AACGACGTCTTC…5’ 5’-TTGCTGCA GAAG…-3’ + ACGTCTTC…5’ 3’-AACG blunt end or flush end Make a simple double-stranded cut in the middle of the recognition sequence 5’-CCCGGG…-3’ 3’-GGGCCC…5’ 5’-CCC GGG…-3’ + 3’-GGG CCC…5’ Sma I *The ligation efficiency between the blunt ends is not as high as that of the stickly ends. Naming of RE REs are usually named after the bacterium from which they are isolated. Escherichia coli RY13 I EcoR I The genus name of bacteria The order of the RE found in bacteria The strain name of bacteria The species name of bacteria The requirements for the gene cloning (1) The target genes (2) The vectors (3) The tool enzymes (4) The host cells The basic process of recombination technique * The preparation of target DNA * The selection and preparation of vectors * The ligation of DNA fragments in vitro * Foreign DNA be transported into host cells * The screening and identifying of target DNA 1. The preparation of target DNA (1)To prepare from genomic library genomic library contains a comprehensive DNA fragments from genomic DNA cut by the specific RE. During the construction of the genomic library, the DNA fragments and their vectors are ligated, and then introduced into the recipient cells. It represents whole DNA sequence of a genome (2) To prepare from cDNA library or cDNA Extracting total mRNA Ligation Reverse transcription introduction It represents the population of mRNAs coding for gene and protein expression (3) To prepare the gene fragment with other methods 1) PCR amplification 2) To synthesize the DNA fragment by chemical method it is typically used for those of the small biologically active peptides 2. The selection and preparation of vectors Plasmid λ phage < 10 kb < 22 kb Capacity of cloning cosmid 40~50 kb M13 phage < 1 kb gDNA library - + + - cDNA library + + - - Subcloning + - - + Sequencing + + - + E coli expression + + - - 3. Construction of Recombinant Molecules Both purified DNA fragments and vectors are digested with the same restriction enzyme to give complementary cohesive ends •Analyzing the result of restriction endonuclease cleavage Separation of molecules by gel electrophoresis Visualizing DNA molecules in a gel (EB staining) Comparison with size markers •Ligated by T4 ligase to recombinant molecules joining together of the vector molecules and DNA to be cloned The enzyme that catalyses the reaction is called DNA ligase, which purified from E.coli bacteria that have been infected with T4 phage (1) Sticky-ended ligation 3’ GAATTC CTTAAG G CTTAA EcoR Ⅰ 5’ 3’ GAATTC CTTAAG GAATTC CTTAAG 5’ 3’ 5’ GAATTC CTTAAG G 3’ CTTAA 5’ ligation GAATTC CTTAAG Bidirection insertions AATTC G 3’ 5’ AATTC 3’ G GAATTC CTTAAG 5’ GAATTC CTTAAG (2) Blunt-ended ligation Target gene Vector Restriction endonucleases Restriction endonucleases T4 DNAligase 15ºC recombinate Self-ligated vector Self-ligated target gene 4. Introduction of DNA into living cells Serves two main purposes: allows a large number of recombinant DNA molecules to be produced from a limited amount of starting material Purification -Methods Transformation Transfection Infection Transformation ----The uptake of DNA by bacterial cells preparation of competent E.coli cells 50 mM CaCl2 is tranditionally used. Another alternative is by electroporation • In recent years, transformation has been extended to include uptake of any DNA molecules by any type of cell Whether the uptake results in a detectable change in the cell Whether the Cells involved is bacterial, fungal, animal or plant Introduction of phage DNA into bacterial cells Two methods: Transfection purified phage DNA, or recombinant phage molecules, is mixed with competent E.coli cells and DNA uptake induced by heat shock Transfection Introduction of phage DNA into bacterial cells Two methods: In vitro packaging single strain system: the defective λphage carries a mutation in the cos sites two strain system: two defective λphage carries a mutation in a gene for one of the components of the phage protein coat Phage infection is visualized as plaques on the agar medium In vitro packaging 5. Screening and Identification of Recombinants The problem of selection A restriction digest of total cell DNA produces not only the fragment carrying the desired gene, but also many other fragments carrying all the other genes Numerous different recombinant DNA molecules are produced A variety of recombinant clones are obtained Target Genes Carried by Plasmid Target Genes Restriction Enzyme DNA Recombination Target Gene Recombination Chromosomal DNA Restriction Enzyme Transformation Host Cells Recombinant Plasmid Transformation 1 plasmid 1 cell Juang RH (2004) BCbasics Amplification and Screening of Target Gene 1 Plating 1 cell line, 1 colony Bacteria Duplication X100 Plasmid Duplication X1,000 Pick the colony containing target gene =100,000 Juang RH (2004) BCbasics There are two basic strategies for obtaining the clone you want Direct selection for the desired gene the only clones that are obtained are clones of the required gene There are two basic strategies for obtaining the clone you want Direct selection for the desired gene the only clones that are obtained are clones of the required gene Identification of the clone from a gene library entails an initial shotgun cloning experiment, to produce a clone library representing all or most of the genes present in the cell, followed by analysis of the individual clones to identify the correct one Correct clone A clone library Direct selection an antibiotic resistance gene Direct selection an antibiotic resistance gene Marker rescue ---by αcomplementation plasmids contain sequence (lacZ) coding for N-terminal amino acids (α fragment) of β–galactosidase Mutant cells contain sequence (lacZ) coding for C-terminal amino acids (ω fragment) of β– galactosidase By αcomplementation The enzymatic activity is dependent on the coexpression of the complete fragments, which can hydrolyzes the specific substrate X-gal (5-bromo-4chloro-3-inolyl-β-D-galactoside) to turn to a blue colored one under the induction of IPTG(isopropyl thiogalactoside) The recombinant molecules have no this enzyme activity because the insertion of target gene into the lacZ region disturbs the expression of αfragment, and therefor, the colour of the recombinant molecule containing the colony is white. Based on this blue-white colony screening α-mutual complement screening Multiple cloning sites Cleavage N end Ampr Ampr The sequence coding the N end fragment of β-galactosidase promoter External DNA Cleavage N end transformation transformation Chromosome The sequence coding the C end fragment of βgalactosidase The growth of bacteria on the culture with X-gal Recombinant pUC18 The growth of bacteria on the culture with X-gal The white clone containing the recombinant pUC18 The blue clone containing the pUC18 The blue clone containing the pUC18 White clone contains the recombinant, but blue clone not contain recombinant Direct selection an antibiotic resistance gene Marker rescue ---by αcomplementation Colony/plaque in situ hybridization is used for positive colony screening Colony Is Screened by Hybridization with Probe Colony hybridization Transferring … Collect filter paper Dissolve cell Autoradiography DNA denatured Add probe Juang RH (2004) BCbasics Cover with filter paper Direct selection an antibiotic resistance gene Marker rescue ---by αcomplementation Colony/plaque in situ hybridization is used for positive colony screening Immunological Technology Methods for clone identification Colony PCR Enzyme digestion nucleic acid hybridization DNA sequencing Using PCR to detect gene targeting events Identification with restriction enzymes M 1 2 3 4 5 6 7 8 9 10 M 1 2 3 4 5 6 7 8 9 10 1kb Figure2a. The clones of positive plasmid M----1Kb DNA ladder 1∽10, positive plasmid A. before cut with RE Figure2b.The positive plasmids after cutted by EcoR I M----1Kb DNA ladder 1∽10, positive plasmids cutted by EcoR B. after cut with RE To identify the target gene band after cutted by RE The basic process of recombination technique * The preparation of target DNA * The selection and preparation of vectors * The ligation of DNA fragments in vitro * Foreign DNA be transported into host cells * The screening and identifying of target DNA 6. Expression of the cloned gene Different vectors are selected for cloning cloning vectors are used for replicating and amplifying genes Expression vector are applied to express the gene product Cloning vector Antibiotics resistance, MCS and screening Expression vector Antibiotics resistance, MCS and screening Contains regulatory sequences for transcription and translation, eg; promoter, SD sequence for 16s rRNA binding, and a terminator which is ρfactor independent Expression system Prokaryote expression system E.coli (most popular) Its easy culture, fast proliferation, low expense, large scale production Lack of the processing capability after transcription and translation Notes during expression Infusion proteins maybe formed when other sequences coding amino acids linked to a target gene are co-expressed together with target proteins Purified by affinity chromatography, followed by the unnsecesary peptides cut out eukaryote expression system Mammalian cells are stable and repeatable can process hnRNA to become mature mRNA, as well as the post-translation modifications Application of Recombination DNA Technology DNA Recombination Medical Production 产 品 功 能 组织胞浆素原激活剂 抗凝 血液因子VIII 促进凝血 颗粒细胞-巨噬细胞集落剌激因子 剌激白细胞生成 促红细胞生成素 剌激白细胞生成 生长因子(bFGF, EGF) 刺激细胞生长与分化 生长素 治疗侏儒症 胰岛素 治疗糖尿病 干扰素( 1b, 2a, 2b, ) 抗病毒感染及某些肿瘤 白细胞介素 激活、剌激各类白细胞 超氧化物歧化酶 抗组织损伤 单克隆抗体 利用其结合特异性进行诊断试验、肿 瘤导向治疗 乙肝疫苗(CHO, 酵母) 预防乙肝 口服重组B亚单位菌体霍乱菌苗 预防霍乱 目录 Gene diagnosis It is recognized that the abnormal structure and expression of the gene are involved in the pathogenesis of diseases Gene diagnosis is the detection of the abnormalities of the candidate genes by ways of molecular biology and molecular genetics For examples: Sickle cell anemia belongs to gene point mutation The 6th codon GAG was changed to GTG Glu is changed to Val Abolish an MstII restriction site which spans codons 5-7 Sequencing HbS proteins revealed a single change: Glu6Val in the β chain. Fiber formation (R) at low [O2] causes sickling of RBCs (center). Restriction mapping analysis of sickle cell anemia MstⅡrestriction site (GCTNAGG) 5´ 3´ 1.15kb Normal gene × 5´ 3´ 1.35kb Mutation gene 镰状红细胞贫血患者基因组的限制性酶切分析 ﹣ 1.35kb 1.15kb 0.2kb + Normal Carrier Sickle cell homozygote Gene Therapy Gene therapy is the way to transfer genetic material which exerts the biological function into the cells of patients to treat the disease Genetic material: normal gene, recombinant DNA, RNA, synthetic oligonucletides They may integrate into the chromosome or express separately The Strategies and technologies of gene therapy Gene correction The abnormal bases of a gene are corrected Gene replacement The defected gene is replaced by the normal one which can integrate into the chromosomes by homologous recombination or remain extrachromosomal The Strategies and technologies of gene therapy Gene augmentation The target gene is introduced to the defected cells or other cells Gene inactivation the expression of the gene is intervened to block or inhibit the inappropriate genes in vivo on both transcriptional and translational level Applications of Gene Therapy The first apparently successful application was initiated on Sep. 14, 1990 for ADA deficiency Results in a lymphopenic form of SCID that is fatal in early childhood. ADA SCID (Severe Combined Immunodeficiency Diseases ) Autosomal recessive disorder ADA = adenosine deaminase (an enzyme of purine metabolism) ADA is an important enzyme in the purine catabolic pathway, catalyzing the irreversible deamination of adenosine to inosine. SCID with ADA Deficiency ADENOSINE Purine Catabolism Pathway Adenosine Deaminase INOSINE GUANOSINE Purine Nucleoside Phosphorylase Purine Nucleoside Phosphorylase GUANINE HYPOXANTHINE Xanthine Oxidase XANTHINE Xanthine Oxidase URIC ACID Guanase SCID with ADA Deficiency The enzyme deficiency inhibits the normal catabolism of purines. Results in the accumulation of metabolic substrates that are toxic to lymphocytes, particularly in the inhibition of lymphocyte function. GENE THERAPY IN ADA SCID ADA deficiency was the first disorder to be treated by gene therapy (Bordignon et al 1995) The initial targets for genetic manipulation were bone marrow (BM) stem cells and peripheral blood lymphocytes (PBLs) GENE THERAPY IN ADA SCID Vectors expressing human ADAcDNA (1.5 kbp) with their own promoters were transfected into BM stem cells and PBLs in vitro 6 months after gene therapy ended, vectorderived DNA was found in the PBLs The Cloning Procedure Used for Creating Dolly