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Laboratory of Molecular Genetics, KNU Gene Cloning Laboratory of Molecular Genetics, KNU Cloning - a definition • From the Greek - klon, a twig • An aggregate of the asexually produced progeny of an individual;a group of replicas of all or part of a macromolecule (such as DNA or an antibody) • An individual grown from a single somatic cell of its parent & genetically identical to it • Clone: a collection of molecules or cells, all identical to an original molecule or cell Laboratory of Molecular Genetics, KNU DNA technology (DNA 조작기술) Joshua Ledergerg & Edward Tatum, 1946 두 종류의 다른 유전자를 가진 대장균 사이에 유전자의 재조합이 일어나 새로운 대장균이 만 들어 지는 것을 발견했다. 이 원리를 바탕으로 1970년대에 DNA재조합 기술이 발전하게 되 었다. Laboratory of Molecular Genetics, KNU 원핵생물에서 DNA의 이동방법 1. 형질전환 (Transformation) 세포 주변에 있는 유전물질을 받아 들이는 방법 (Frederick Griffith 1920) 2. 형질도입 (Transduction) 세균의 유전자를 박테리오 파지가 전달 3. 접합 (Conjugaton) 두 개의 세포사이에 접합을 통한 DNA 이동 Laboratory of Molecular Genetics, KNU The host cell : Escherichia Coli DNA 증식을 위해 transformation 중요 Transformation을 위해 요구되는 요소 - gene 도입을 위한 적당한 숙주 - 숙주 안으로 gene을 도입할 운반체 - gene을 받아들인 숙주 선별할 수단 Bacterium E. coli - 가장 넓게 사용 ; simple, genetic environment 잘 알려짐 - genome 완전히 분석됨 - genetic code 보편적이기 때문에 다른 생물의 외래 DNA 받아들임 → DNA의 구성, 구조, 기본 mechanism 동일하므로 복제 가능 - 빠르게 분열하고, cell 분열할 때마다 도입된 DNA도 복제 - culture medium에서 37℃일 때 최고 성장 - bacterial growth Laboratory of Molecular Genetics, KNU Proposed molecular mechanism of DNA transformation of E. coli 0 ℃ 처리하여 세포막 고형화, charged phosphate stabilizing - Transformation solution속의 양이온들이 phosphate group과 complex 이룸 → (-) charge 가리므로 DNA 분자 이동 가능 - Heat shock → 세포막의 열적 불균형 형성하여 adhesion zone 통한 DNA pumping 도움 Laboratory of Molecular Genetics, KNU The Boyer-Cohen- Chang experiment. 1973 Proof that The Boyer-Cohen- Chang experiment created a recombinant DNA molecule Laboratory of Molecular Genetics, KNU DNA Recombination inserting new genes into plasmids - gene cloning technology ; cut and past DNA fragment - plasmid vector는 cloning site(제한효소 인식 서열) 포함 ; 원형의 plasmid 절단하여 open, DNA 삽입 가능 - 제한효소 → sticky end 형성 ; 보완적인 다른 fragment와 수소 결합 형성하므로 DNA ligase를 위해 충분한 시간 동안 DNA fragment 잡아둠 - DNA ligase ; 인접한 nucleotide 사이에 phosphodiester 결합 재형성 → stable double helix Laboratory of Molecular Genetics, KNU Laboratory of Molecular Genetics, KNU Restriction endonucleases cut DNA - DNA의 특정 sequence 인지, 절단(break phosphodiester bond) 발견과정 -1950s; bacteria에서 원시적인 immune system 발견 -1960s; enzyme system (E. coli 추출물) 발견 → self DNA 보호, 외래 DNA 인지- 절단 modification activity (methylation) -1970s; New restriction endonuclease발견 → Hind Ⅲ (haemophilus influenzae 에서 발견) → modification activity 없음, 인식자리 안의 정확한 지점 절단 Laboratory of Molecular Genetics, KNU Restriction endonucleases Three major class - type Ⅰ, Ⅲ ; restriction and modification activity , 인식자리 밖 절단, ATP를 에너지원으로 사용 → 예측 불가능, ATP 요구성 때문에 사용 안 함 - type Ⅱ ; restriction activity만 있음, ATP필요 없음, Mg2+ 필요, 인식자리 안이나 인접부위 예측 가능하게 절단 → DNA 조작에 이상적 절단방법 - middle of the site → blunt end - 3’ of center, 5’ of center → sticky end Frequency of cutting ; 제한효소가 인지하는 sequence의 길이에 의존 Restriction map ; 제한효소에 의해 절단한 DNA fragment의 크기 비교 → genetic map과 연관 Laboratory of Molecular Genetics, KNU Restriction enzymes cleave DNA at a specific sequence Laboratory of Molecular Genetics, KNU Molecular detail of EcoR1 restrictionmodification Laboratory of Molecular Genetics, KNU Properties of restriction enzymes-2 HaeIII Haemophilus aegiptius GG/CC Blunt cut Sau3A Staphylococcus aureus /GATC 5’-overhang HhaI Haemophilus haemolyticus GCG/C 3’-overhang SmaI Serratia marcescens CCC / GGG Blunt cut EcoRI Escherichia coli RY13 G / AATTC 5’-overhang PstI Providencia Stuartii CTGCA / G 3’-overhang HaeII Haemophilus aegiptius RGCGC / Y Ambiguous sequence NotI Nocardia otitidis GC / GGCCGC 8 nt sequence Laboratory of Molecular Genetics, KNU Laboratory of Molecular Genetics, KNU Laboratory of Molecular Genetics, KNU Laboratory of Molecular Genetics, KNU Plasmid selection selectable markers - plasmid 삽입된 cell과 삽입되지 않는 cell 구별을 위해 사용 - antibiotic resistance 이용 ; plasmid 삽입된 cell만 항생제 함유 배지에서 생존 - antibiotic resistance → chemical modification 통해서 target antibiotics inactivation → 세포막을 통한 antibiotics transport 방해 Laboratory of Molecular Genetics, KNU 플라스미드를 이용한 cloning 1.미생물로부터 플라스미드를 분리한다. 2. 동물, 식물로부터 특정한 유전자가 포함된 DNA를 분리한다. 3. 분리한 유전자를 포함하고 있는 DNA조각을 플라스미드에 삽입하여 재 조합 DNA를 만든다. 4. 박테리아 세포에 재조합 플라스미드 를 넣어 형질전환을 한다. 5. 재조합 박테리아 클론이 확보 사용된 다. Laboratory of Molecular Genetics, KNU Transformation E. Coli ; CaCl2 + heat shock(42℃) 조건에서 형질전환 일어남 다른 이온(Mg2+, Mn2+,Ba2+ 등)도 사용 ; mixture of positive ion 사용시 → 효율 증가 DNA size 와 conformation이 형질전환 효율에 영향 A subset of cell에 제한 받음 → plasmid 수 증가해도 형질 전환된 cell 수 변화 없음 E. coli가 DNA 받아들이는 정확한 메커니즘 밝혀지지 않음 → adhesion zone 가설 ; 세포막의 adhesion zone 에서 channel 형성 단점; Large DNA는 성공적으로 형질전환 되기 힘듦 Laboratory of Molecular Genetics, KNU Directional cloning Laboratory of Molecular Genetics, KNU Genomic library (유전자 도서관) 무차별 유전자 클로닝 방법 1. 제한효소를 이용하여 DNA를 수천 조각으로 절단 2. 각 DNA조작은 서로 다른 벡터 분자에 실려 박테리아 세포에 형질 전환 3. 수많은 종류의 박테리아 클론 을 genomic library 라 함 Laboratory of Molecular Genetics, KNU The plasmid vector propagation of plasmids - bacterial cell의 빠른 증식 능을 이용하여 특정 gene을 증폭 시킬 때 plasmid 이용 (host cell division시에 plasmid duplication) - origin of replication(ori) sequence 필요 → host cell 안에서 복제 가능하게 함 - 복제 조절에 따라 2 group으로 구분 → strigent control ; bacterial cell 분열에 조절 받음(1개씩 replication) → relaxed ; bacterial cell 과 자율적(cell당 수 백개의 copy 축적) Laboratory of Molecular Genetics, KNU pBR322 Laboratory of Molecular Genetics, KNU pUC19 Laboratory of Molecular Genetics, KNU pUC19 Laboratory of Molecular Genetics, KNU GST-Vector (pGEX 6p, T7) Laboratory of Molecular Genetics, KNU Laboratory of Molecular Genetics, KNU Eukaryotic expression vector Laboratory of Molecular Genetics, KNU Isolation of recombinant plasmids 1.E.coli을 EDTA, Glucose 섞인 buffer로 현탁 2. SDS, NaOH Mixture 첨가 → cell lysis, DNA denature 3. potassium acetate, acetic acid 첨가 → neutralization 4. 상층액에 ethanol 이나 isopropanol 첨가 → plasmid DNA 침전 5. pellet = clean plasmid DNA 6. 전기 영동 하여 재조합 확인 Laboratory of Molecular Genetics, KNU Size separation of DNA fragments by electrophoresis in agarose gels DNA is negatively charged due to phosphates on its surface. As a result, it moves towards the positive pole. Laboratory of Molecular Genetics, KNU Laboratory of Molecular Genetics, KNU Gene Therapy It is a technique for correcting defective genes that are responsible for disease development There are four approaches: 1. A normal gene inserted to compensate for a nonfunctional gene. 2. An abnormal gene traded for a normal gene 3. An abnormal gene repaired through selective reverse mutation 4. Change the regulation of gene pairs Laboratory of Molecular Genetics, KNU How It Works A vector delivers the therapeutic gene into a patient’s target cell The target cells become infected with the viral vector The vector’s genetic material is inserted into the target cell Functional proteins are created from the therapeutic gene causing the cell to return to a normal state Laboratory of Molecular Genetics, KNU The First Case The first gene therapy was performed on September 14th, 1990 Ashanti DeSilva was treated for SCID Sever combined immunodeficiency Doctors removed her white blood cells, inserted the missing gene into the WBC, and then put them back into her blood stream. This strengthened her immune system Only worked for a few months Laboratory of Molecular Genetics, KNU http://encarta.msn.com/media_461561269/Gene_Therapy.html Laboratory of Molecular Genetics, KNU Viruses Replicate by inserting their DNA into a host cell Gene therapy can use this to insert genes that encode for a desired protein to create the desired trait Four different types Laboratory of Molecular Genetics, KNU Retroviruses Created double stranded DNA copies from RNA genome The retrovirus goes through reverse transcription using reverse transcriptase and RNA the double stranded viral genome integrates into the human genome using integrase integrase inserts the gene anywhere because it has no specific site May cause insertional mutagenesis One gene disrupts another gene’s code (disrupted cell division causes cancer from uncontrolled cell division) vectors used are derived from the human immunodeficiency virus (HIV) and are being evaluated for safety Laboratory of Molecular Genetics, KNU Adenoviruses Are double stranded DNA genome that cause respiratory, intestinal, and eye infections in humans The inserted DNA is not incorporate into genome Not replicated though Has to be reinserted when more cells divide Ex. Common cold Laboratory of Molecular Genetics, KNU Adenovirus cont. Laboratory of Molecular Genetics, KNU Adeno-associated Viruses Adeno-associated Virus- small, single stranded DNA that insert genetic material at a specific point on chromosome 19 From parvovirus family- causes no known disease and doesn't trigger patient immune response. Low information capacity gene is always "on" so the protein is always being expressed, possibly even in instances when it isn't needed. hemophilia treatments, for example, a gene-carrying vector could be injected into a muscle, prompting the muscle cells to produce Factor IX and thus prevent bleeding. Study by Wilson and Kathy High (University of Pennsylvania), patients have not needed Factor IX injections for more than a year Laboratory of Molecular Genetics, KNU Konckout Mice Transgenic Mouse: Generic term for an engineered mouse that has a normal DNA sequence for a gene replaced by an engineered sequence or a sequence from another organism. Knockout Mouse: A transgenic mouse in which the normal gene is missing or engineered so that is not transcribed or translated. “Knocks out” that gene. Knockin Mouse: A transgenic mouse in which the engineered “transgene” is subtly manipulated to: (A) alter the function of the gene (e.g., replace one amino acid with another in a site to determine if that site is essential for the protein’s function); (B) change transcription rate to overproduce or underproduce the gene product; or (C) create a fluorescent gene product to map its distribution in tissue. Conditional Knockout (Knockin) Mouse: A transgenic mouse in which the transgene is knocked out (or in) in specific tissues, at a specific developmental stage, or in response to an exogenous substance (e.g., an antibiotic). Laboratory of Molecular Genetics, KNU Transgenic Organisms General Outline: • Infect blastocyst cells/sperm with viral vector with the gene of interest. • Hope that in some cells homologous recombination will insert the DNA section of interest into the target cell’s chromosome. •Select chimeric organisms. •Breed until the transformed DNA is found in a germ line. Laboratory of Molecular Genetics, KNU (1) Get the nucleotide sequence of the gene of interest. Including upstream and downstream nucleotides. agctta tcgaat Gene of Interest cgatc gctag Downstream DNA (unique) to the gene, usually > 1 kb Upstream DNA (unique to the gene), usually > 1kb (2) Construct the desired DNA sequence (i.e., the transgene), adding a gene for antibiotic resistance, but keeping the upstream and the downstream nucleotides. agctta tcgaat Desired Gene Antibiotic Resistance Gene cgatc gctag Laboratory of Molecular Genetics, KNU (3) Micropipette embryonic stem cells from the inner cell mass of a blastocyst (i.e. early mouse embryo) in a strain with a physically recognizable phenotype (e.g., pigmented). (4) Culture the cells with many copies of the manufactured transgenic DNA complex. Short bursts of an electrical current allow the DNA to pass through the plasma membrane into the cell (electroporation). Laboratory of Molecular Genetics, KNU (5) Cells will divide in culture and some of them will incorporate the transgenic DNA strand into the chromosome (homologous recombination). After a sufficient number of cell divisions, add the antibiotic. This will preferentially kill those stem cells that have not incorporated the transgenic strand (black dots), giving a good harvest of those that have incorporated the strand (red dots). + antibiotic (6) Insert the stem cells into the blatocyst of a mouse with a different genetic background trait (e.g., an albino if the original stem cells came from a pigmented mouse). Laboratory of Molecular Genetics, KNU (7) Implant the new blastocysts into a pseudopregnant female with a visible phenotype different from the blastocyst phenotype (e.g., albino if the blastocyst is pigmented). (8) Offspring that have pigmented sections are chimeras that have incorporated the transgenic sequence into their cell lines. Select them for further breeding. Laboratory of Molecular Genetics, KNU (9) Keep breeding the offspring of the chimeras until some fully pigmented mice are born. A fully pigmented mouse means that the transgenic germline generated one of the gametes that resulted in that mouse. Genotype the mouse to determine the genotype at the desired locus and the insertion point(s). (Most will be heterozygotes for the wild type allele and the transgenic allele). (10) Mate two heterozygotes and genotype their offspring. This will give all three genotypes--wild type homozygotes, heterozygotes, and transgenic homozygotes. (11) Compare the three genotypes on the phenotype of interest. Laboratory of Molecular Genetics, KNU Laboratory of Molecular Genetics, KNU