Room: 7331

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318 423 ????????????????
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?????????????? ?????????????????? Room
7331 Email sthanaset_at_kku.ac.th
References 1. Kingsman SM and Kingsman AJ.
Genetic Engineering. Oxford Alden Press,
Blackwell Scientific Publications, 1990. 2.Joyner
AL. Gene Targeting A Practical Approach. 2nd
edition. New York Oxford University Press Inc.,
2000. 3.Old RW and Primrose SB. Principles of
Gene Manipulation An Introduction to Genetic
Engineering. 5th edition. Oxford the Alden Press
Limited, Blackwell Scientific Publications,
1994. 4.Primrose SB and Twyman RM. Principles of
Gene Manipulation and Genomics. 7th edition.
Oxford Blackwell Publishing, 2006. 5.Http//www.v
oice.buz.org/genetic_engineering/ethicsandge.html
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????????????????????
??????? Gene Transfer into Animal Cells -
Animal cells - Gene transfer vectors -
Expression vectors - Techniques for
transferring DNA into animal cells Gene
Transfer into Whole Animal - Gene manipulation
in Mouse - Gene manipulation in Drosophila
- Gene manipulation in Xenopus laevis Ethical
Concerns on Genetic Engineering in Animal
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????????????????????
????????????? (Genetic Engineering) ???????
????????? ????????????????? (gene)
??????????????????????????????????????? ??????????
???????????? ???????????????????????????????
???????????? ??????????????? ????????????????????
???????????????????? ??????????????????????
???????????????????? (Genetic Engineering in
Animal) ???????????????????????????????????
????? ????????????????????? ??????????????????????
1. Gene Transfer into Animal Cells 2. Gene
Transfer into Whole Animals
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Gene Transfer into Animal Cells
Animal Cells - ???????????? (model)
???????????????????????????? ???????? ????????
???? model ???????????????????, ???????????????
??? ????????????????????? ???????
Animal Cell Culture 1. Primary Cell Culture
- have a finite life span (10-20 weeks 30-60
cell divisions) - contact
inhibition 2. Continuous Cell Line (Immortalized
Cells) - propagate indefinitely -
usually aneuploid - reduced nutritional
requirements - divide rapidly - lose
contact inhibition
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Note Specialized cells are unable to grow in
cell culture ? loss of proliferative capacity
terminal differentiation

• - use immortalized derivatives
• Immortalize cells by..
• 1. infect cells with tumor virus, eg. SV40
• 2. transform with an oncogene

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Basic expression vector
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??????????? DNA ????????????????????????????????
(mammalian cells) Transfection 1.Non Viral
Transfection System (physical transfection,
chemical transfection, bactofection) 2.Viral-based
Transfection System (Transduction)
Non Viral Transfection System
1. Calcium phosphate precipitation (chemical
transfection) -???????? Bacchetti S. and
Graham L. (1977) -??? DNA ??? calcium
chloride ?? phosphate buffer
???????????????????????????? DNA-
CaPO4, DNA ??????????????? endocytosis
2. DEAE-Dextran (chemical transfection)
-facilitate DNA binding to cell membrane
-DNA ??????????????? endocytosis
-transient transfection
3. Cationic Liposome-mediated transfection
(chemical transfection) -Lipofectamine
-Fugene6
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Endocytosis
Endocytosis

• Endocytosis
• Fusion

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4. Linear cationic polyethylenimine (chemical
transfection)
-jetPEI
PEI Polyethylenimine
Endocytosis
-??????????? non attached cells
Biomol company
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5. Electroporation/electropermeabilization
(Physical transfection) -???????????
(pore) ?????????????????????????????????
-pore ????????????????????????????????????????????
????????????????????? (dielectric strength)
-??????????????????????????
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6. Nucleofection ( electroporation) (physical
transfection) - ?????????? 1.
Nucleofector-set optimum electrical parameter
for specific cell type 2. Nucleofector
solution-cell type specific
3.
1.
2.
4.
Amaxa, a Lonza company
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7. Magnetofection (physical transfection)
-DNA magnetic particles -----gt apply
magnetic force -Poly MAG, Combi MAG
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8. Microinjection (physical transfection)
-inject DNA directly into nucleus of cells using
glass micropipettes -Cells are
grown on glass slides -500-1000 cells/hr
-success 50-100
9. Protoplast fusion (Bactofection)
Bacterial cells containing plasmids of interest
Fuse bacterial protoplast to
mammalian cells by using polyethylene
glycol - contaminated bacteria are killed by
antibiotic in the medium - Not widely used
Treat with lysozyme-remove cell wall
(generate protoplast)
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Viral-based Transfection System
- Viruses intracellular parasites (cell type
specific) transfect their own
DNA/RNA into the host cell ---gt
produce new viral particles
- Increased transfection efficiency - Potentially
infectious particles
1. Retrovirus Vector
- Virus ssRNA genome

• Moloney murine leukemia virus
• (Mo-MLV)

- 10 kb

• At least 3 genes
• gag -coding for core proteins
• pol -coding for Reverse transcriptase
• env -coding for viral envelope protein

- LTR long terminal repeat
(promoter/enhancer/integration)
- infect only dividing cells
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http//biology.kenyon.edu/slonc/gene-web/Lentivira
l/HIVvector.jpg
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2. Lentivirus Vector
- Virus a subclass of retrovirus

• Able to infect both proliferating
  non-proliferating cells

• More complicated than simple retrovirus
  (..contains additional
• 6 genes tat, rev, vpr,vpu,nef,vif)

3. Adenovirus Vector

• Adenoviruses
• non-enveloped viruses
• linear dsDNA genome
• gt40 serotype strains
• cause benign respiratory
• tract infection in human
• does not integrate into host
• genome
• 35 kb (upto 30 kb can be
• replaced with foreign DNA)
• very efficient viral vectors
• ITRInverted Terminal
• Repeat

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4. Adeno-associated virus (AAV) Vector

• AAV
• non-pathogenic human parvoviruses
• linear ssDNA genome 5kb
• (insert not larger than 4.7 kb)
• dependent on helper virus for
• replication (..require Adenoviruses
• or Herpes viruses)
• integrate into the host genome
• 145 bp Terminal Repeat (TR)
• very efficient viral vectors
• ITRInverted Terminal Repeat

• AAV vector
• cumbersome method, low yield,
• contamination

5. Herpes Simplex virus (HSV) Vector

• HSV-1
• Human Neurotropic virus
• Vector for gene transfer to the
• nervous system
• linear dsDNA , 152 kb (40-50 kb
• foreign DNA can be inserted)

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Promoter control of foreign DNA
-Most vectors use CMV promoter -
smaller, stronger, better understood than most
human promoter sequences
Stable and transient transfection
-Transient foreign DNA is lost at the later
stage when the cells
undergo mitosis -Stable foreign DNA is inserted
into the nuclear genome
(Integration randomly
specifically (homologous
recombination)
---gt knock-in/knock-out checked
by co-transfected gene (selectable marker) whose
its product can neutralize toxin
---gt Neomycin resistant gene
---Geneticin (G418)
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Gene Transfer into Whole Animal
- Cells in culture are unlikely to mimic all
aspects of behaviour of the same cells in the
animal - Transferring genes into an intact animal
to follow gene expression during development and
differentiation
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Major experimental organisms - Mouse - Drosophila
melanogaster - Xenopus laevis
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Gene transfer into mouse

• Microinject DNA
• into fertilized eggs

http//dels.nas.edu/ilar_n/ilarjournal/38_3/38_3Tr
ansgenic.shtml
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http//www.scq.ubc.ca/wp-content/ pronuclearmicroi
njection.gif
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2. Viral infection
- co-cultivate 4-8 cell pre-embryos with cells
producing virus (infectious recombinant
retroviral vectors) - inject virus into the
blastocyst in vitro
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Cell transfer into embryos
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Nuclear transfer technology
- Nuclear transfer technology can be used to
clone animal
http//www.millerandlevine.com/cloning/dolly-fig-1
3-13.jpg
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Ethical Concerns on Genetic Engineering in Animal
http//www.boyd-group.demon.co.uk/genmod.htm
1. Fundamental moral objections (i) Objections to
the use of animals in general - object to any
experiment which causes animals pain and
suffering - object to all human uses of animals,
whether or not pain and suffering is
involved (ii) Objections to the genetic
modification of animals in particular -
objecting that genetic engineering is
'unnatural', 'playing God', and that it
'debases animals' by treating them as
'commodities - objections to the creation of
animal strains which suffer throughout their
lives because of their genetic
make-up
2. Concerns about the consequences of genetic
modification of animals (i) concern about
consequences for the welfare of modified animals
(ii) concern about the harms caused during
the production of modified animals (iii)
concern about the hazards which modified
organisms might pose to human and animal
health and to the environment
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