Transcript Laboratory of Molecular Genetics, KNU

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




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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