Making Transgenic Plants and Animals

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Making Transgenic Plants and Animals

• Why?
• Study gene function and regulation
• Making new organismic tools for other fields of
  research
• Curing genetic diseases in people
• Improving agriculture and related raw materials
• New sources of bioengineered drugs (use plants
  instead of animals of bacteria)

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Transgenic Mice
The organism of choice for mammalian genetic
engineers. - small - hardy - short life
cycle - genetics possible - alot of useful
strains and tools
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DNA Integration

• Can occur by homologous (H) or non- homologous
  (N-H) recombination
• Frequency of N-H gtgt H (by 5000- fold)
• If you want H integrants, which you need for
  knock-outs, you must have a selection scheme for
  those

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Vector with a transgene
tk1 tk2 - Herpes Simplex Virus thymidine
kinase genes (makes cells susceptible to
gancyclovir) Neo - neomycin resistance
gene Homologous regions - homologous to
chromosomal target Transgene foreign gene
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Example of what happens with N-H recombination
Transformed cells are neo-resistant but
gancyclovir sensitive.
homol--gt
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What happens with HR
If DNA goes in by HR, transformed cells are both
neo-resistant and gancyclovir-resistant! Use
double-selection to get only those cells with a
homologous integration event (others are killed).
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• To knock-out a gene
• Insert neo gene into the target gene.
• Transform KO plasmid into embryonic stem cells.
• Perform double-selection to get cells with the
  homologous integration (neo gangcyclovir
  resistant).
• Inject cells with the knocked-out gene into a
  blastocyst.

1.
KO
KO
2,3.
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How to make a transgenic mouse
With DNA
(mouse)
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Chimeric mouse
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• If the recipient stem cells are from a brown
• mouse, and the transgenic cells are
• injected into a black (female) mouse, chimeras
  are
• easily identified by their Brown/Black
• phenotype.
• To get a completely transgenic KO mouse (where
  all cells have KO gene), mate the chimera with a
  black mouse. Some of the progeny will be brown
  (its dominant), because some of the germ line
  cells will be from the KO cells. ½ the brown mice
  will have the transgene KO, because the paternal
  germ-line cell was probably heterozygous.
• To get a homozygous KO mouse (both chromosomes
  have the KO transgene), cross two brown
  transgenic heterozygotes. 1/4 will be homozygous
  at the transgene locus.

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Not necessarily 31
Fig. 5.40 (from 5.7)
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Arteries from a mouse with a KO of the
low-density lipoprotein receptor-related protein
(LRP).
Mouse is actually a double-KO, with the LDL
receptor also knocked out. This was generated by
crossing the LRP-KO mouse with a LDL receptor-KO
mouse. The LDL receptor-KO makes mice
particularly susceptible to cholesterol feeding.
Science 300, 329 (2003)
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Gene therapy in humans presents some formidable
problems

• If you could introduce the gene in early
  development (e.g., eggs? or blastocyst) might
  cure (or partially cure) many diseases
• How to fix them later, as a child, adolescent,
  adult, etc.?
• Transgenic technology stem cell technology
  many interesting possibilities

users.rcn.com/jkimball.ma.ultranet/BiologyPages/
T/TransgenicAnimals.html
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Transgenic plants
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• Two main ways of getting DNA into plant
  chromosomes
• Agrobacterium- mediated gene transfer
• Direct gene transfer

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Transgenics Direct DNA Transfer

• Introduce naked DNA into cells (plant or animal)
• Can assay expression of the gene immediately, or
  select cells that are permanently transformed
  cells
• DNA introduction methods
• Chemical
• Microinjection
• Electroporation
• Particle bombardment (Biolistics)

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Chemically-induced transformation

• Usually use on cells without walls
• Multiple protocols
• put DNA inside artificial membranes (liposomes),
  they will fuse with plasma membrane
• Bind DNA with polycations to neutralize charge,
  some cells endocytose the complex
• Combine (2) and (1)

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needle
Microinjection of DNA into the pronucleus of a
newly fertilized egg. Injection is usually into
the sperms pronucleus because its larger.
1-2 picoliter vol is injected. 5-40 of
animals will contain transgene.
From Primrose, Molec. Biotechnology
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Electroporation

• Use on cells without walls (plant protoplasts or
  animal cells )
• Used on monocots (maize, rice, etc.)
• High-voltage pulses cause pores to form
  transiently in cell membrane, DNA slips in
• Drawback - its more cumbersome to regenerate
  plants from single protoplasts than from the
  tissue transformations with Agrobacterium

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Particle Bombardment (Biolistics)

• Less limitations than electroporation
• Can use on cells with walls, or essentially any
  tissue
• Can transform organelles
• Method
• Precipitate DNA onto small tungsten or gold
  particles.
• Accelerate particles to high speeds to penetrate
  cells and tissues.
• Perform selective growth and regeneration of
  transgenic plants as described for Agro-mediated
  transformation.

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The Helium Gas Gun Circa 2000
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Repairing an organellar gene 1 x 107 cells of
a mutant of Chlamydomonas that had a deletion in
the atpB gene for photosynthesis was bombarded
with the intact atpB gene. Then, the cells were
transferred to minimal medium so that only
photosynthetically competent cells could grow.
Control plate cells were shot with tungsten
particles without DNA
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Agrobacterium tumefaciens, a natural plant
genetic engineer

• Soil bacterium, related to Rhizobium
• causes crown galls (tumors) on many dicots
• Infection occurs at wound sites

Infected Tobacco w/teratoma
Brief recitation in Weaver, pp. 85-89
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Crown galls caused by A. tumefaciens on
nightshade.
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Lots of pili
complex bacterium genome has been sequenced 4
chromosomes with 5500 genes
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Agrobacterium infection and tumorigenesis

• Infection occurs only at wound sites
• Involves recognition and chemotaxis of the
  bacterium toward wounded cells
• galls are real tumors, can be removed and grow
  indefinitely without hormones
• genetic information must be transferred to plant
  cells

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Tumor characteristics

• hormone (auxin cytokinin) levels altered,
  explains abnormal growth
• synthesize a unique amino acid, called opine
• octopine and nopaline (derived from arginine)
• agropine (derived from glutamate)
• specific opine depends on the strain of A.
  tumefaciens
• opines are catabolized by the bacterium, which
  can use only the specific opine that it caused
  the plant to produce

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Elucidation of the TIP (tumor-inducing principle)

• It was recognized early that virulent strains
  could be cured of virulence, and that cured
  strains could regain virulence when exposed to
  virulent strains suggested an extra- chromosomal
  element.
• Large plasmids were found in A. tumefaciens and
  their presence correlated with virulence called
  tumor-inducing or Ti plasmids.

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Ti Plasmid

• Large (-200-kb)
• Conjugative
• 10 of plasmid transferred to plant cell after
  infection
• transferred DNA (called T-DNA) integrates
  semi-randomly into nuclear DNA
• Ti plasmid also encodes
• enzymes involved in opine metabolism
• proteins involved in mobilizing T-DNA (Vir genes)

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T-DNA
auxA auxB cyt ocs
LB
RB
LB, RB left and right borders (direct
repeat) auxA auxB enzymes that produce
auxin cyt enzyme that produces cytokinin Ocs
octopine synthase, produces octopine
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Vir (virulent) genes

• Found on the Ti plasmids
• Transfer the T-DNA to plant cell
• acetosyringone (AS) (a flavonoid) released by
  wounded plant cells activates vir genes
• virA,B,C,D,E,F,G (A-E are operons with multiple
  ORFs), span about 30 kb of Ti plasmid

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Vir genes functions (cont.)

• virA - transports AS into bacterium, activates
  virG post-translationally
• virG - promotes transcription of other vir genes
  
• virD2- endonuclease that cuts T-DNA at the
  borders but only on one strand attaches to the
  5' end of the SS
• virE2- DNA-binding protein, binds SS of T- DNA
• virD2 virE2 also help T-DNA get to nucleus in
  plant cell, they have NLSs
• virB - 11 ORFs, helps DNA-protein complex get
  through cell membranes

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From Covey Grierson
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Hypothetical model for virB membrane channel
From P. Zambryski
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• Monocots don't produce AS in response to
  wounding.
• Important Put any DNA between the LB and RB of
  T-DNA it will be transferred to plant cell!

Engineering plants with Agrobacterium Two
problems had to be overcome (1) Ti plasmids
large, difficult to manipulate (2) couldn't
regenerate plants from tumors
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Binary vector system

• Strategy
• Move T-DNA onto a separate, small plasmid
• Remove aux and cyt genes
• Insert selectable marker (drug resistance) gene
  in T-DNA
• Vir genes are retained on a separate plasmid

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Binary vector system (cont.)

• 5. Put foreign gene between T-DNA borders
• 6. Co-transform Agrobacterium with both
  plasmids
• 7. Infect plant with the transformed bacteria
• Leaf-disc transformation common after selection
  and regeneration, get plants with the introduced
  gene in every cell - Transgenic plant

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Binary vector system for Agrobac-terium
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Making a transgenic plant by leaf-disc
transformation with Agro.
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Floral dip method
Female part gets transformed seeds are
heterozygous. You can select homozygous in the
later generations.
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Transgenic Plants In Use or About to be on a
Large Scale

• Herbicide-resistant plants
• Pest-resistant plants
• Vaccine plants (just starting to be used)

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Herbicide-resistant plants

• Resistant to herbicide Round-up (Glyphosate)
• Contain bacterial EPSP synthase
• Advantages better weed control, less tillage
• soybeans, corn, rice, wheat

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Pest-resistant plants

• Resistant to certain insects
• Plants carry gene(s) for Bacillus thuringiensis
  (Bt) toxin
• Advantage less insecticide required, better
  yield
• corn, cotton, potatoes

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Vaccine plants

• cheap vaccine-delivery system
• use plants producing pathogen protein to induce
  immunity
• potatoes, bananas

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Concerns that have been raised about cultivating
and consuming GM crops

• They may be toxic or allergenic.
• They may become established in the wild and
  outcompete other plants.
• They may negatively affect insects or other
  organisms that use crops.
• They may outcross to a nearby wild relative
  spreading the transgene into a wild population.

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References on release of GM crops into the
environment

• Nap et al. (2003) Plant Journal 33, 1-18
• Focuses on current status and regulations
• Conner et al. (2003) Plant Journal 33, 19-46
• Focuses on ecological risk assessment