Gene Manipulation 9

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Gene Manipulation 9

• Transgenic Plants and Animals

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Plants

• Tissue culture
• Culture undifferentiated cells (from cortex,
  meristem) in suitable media
• growth of undifferentiated callus
• grow in suspension, or on agar, ie large and
  small scale
• Callus culture undergoes somatic embryo-genesis
  to give embryoids, which develop into fully
  functional plants.

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Agrobacterium

• Grows on dicotyledons to give crown gall disease,
  a plant tumour.
• Tumour tissue is caused by a plasmid, known as
  Ti.
• 23 kb piece of Ti is found in tumour tissue
• inserted DNA flanked by 25 bp repeats
• Vir genes on Ti plasmid are responsible for the
  transfer process, but are not transferred

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Agrobacterium-2

• Transformed plant tissue produces opines, a
  food source for agrobacterium
• Transformed plant tissue will not regenerate,
  unlike normal callus
• Ti plasmid too large for cloning (too many R.E
  sites, etc)

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Agrobacterium vectors
Vir genes
MCS
neor
Left border
right border
BIN19
pAL4404
Kanr
DNA cloned into MCS, plasmid transformed into
Agrobacterium (select Kanr). Agrobacterium is
mixed with protoplasts, and vir genes transfer
the DNA between borders Select for neomycinr in
plant cells. Kill bugs with tetracycline.
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Monocots

• Agrobacterium only for dicotyledons
• Electroporation of single plant cells
• Select with Neor- place Neor gene in front of
  plant promoter. Regenerate Neor protoplasts into
  whole plants. Many cereals do not regenerate from
  protoplasts/ callus
• Inject DNA directly into developing gamete
• Microprojectiles
• Coat 4mM gold bullets with plasmid DNA. Fire into
  germinal tissue, select seedlings with Neor

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Applications

• Plant gene regulation
• Attach gene regulatory sequences to a reporter
  gene (eg CAT,Luciferase p.312-314), put construct
  into plant, and study expression of CAT, Luc.
• Identify DNA sequences which regulate specific
  genes
• Identify proteins which bind to the DNA sequences

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Applications-2

• High level production of foreign genes in plants,
  eg for production of proteins.
• Improvement of plant growth
• eg insert weedkiller resistance gene. Spray crops
  directly with weedkiller. Kills all weeds, gives
  high yields
• Nitrogen fixation genes (Nif) would allow plants
  to grow in poor soil
• Fungus resistance genes

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Transgenic animals
Allow sperm and ovum to fertilise wait 4 hours.
DNA solution
Female Male pronuclei
Pronuclei fuse development
Approx 3-40 transgenes. DNA incorporates into
genome- often multiple copies of the inserted DNA.
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Transgenic breeding
TT x ww Tw Tw Tw Tw F1 ww Tw Tw TT F2
The transgene is carried as a conventional
mendelian trait. Check for the presence of
homozygotes by southern blotting/ PCR.
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Integration of transgene

• Linear DNA integrates well (not circular)
• DNA integrates at random sites, at variable copy
  number, in head-to-tail arrays
• Expression of DNA is proportional to copy number
• Site of integration effects
• some regions of chromatin are in an inactive or
  active state- this affects trans-cription of
  the transgene in a tissue specific manner.

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Protein production

• Mice are too small for large scale production of
  recombinant proteins
• Use goats, sheep, cattle, etc
• Optimal post-translational modification
• eg milk proteins secreted (via signal peptide)
  into milk. Fuse signal to gene
• harvest from milk. Successful for factor IX,
  yielding mg quantities.
• Great potential, very cheap

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Gene regulation

• Transgenic animals essential where there is no
  suitable cell line model
• b-globin expressed at very high levels in rbc.
  Strict developmental (a, g, d in fetal life) and
  tissue regulation.
• The globin gene cluster contains 5 globin genes
  in 100 kb DNA
• No suitable cell for studying globin transcription

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b-globin

• When the b-globin gene is inserted into
  transgenic mice, with flanking DNA
• transgene expressed at low levels
• position effects variable expression between
  mice, and tissue- specific expression
• expression is not correlated with the copy number
  of the gene
• Regulation is not the same as in vivo

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Globin gene cluster
100kb
e g d b
0kb
Minigene
Site where DNA is very sensitive to nuclease. DNA
normally wrapped up in chromatin wrapper thus
implies the presence of an open region of DNA
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b-globin minigene

• The minigene contains DNA from 60 kb 5 and 20 kb
  3
• Transgenic mice show correct tissue-specific and
  developmental expression
• Equivalent expression to endogenous gene
• Expression is proportional to the number of
  transgenes
• Negligible position effects

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Globin cluster
DNA normally tightly wrapped up.
The sensitive sites of DNA open up a whole region
of DNA in a tissue specific manner.
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Gene function

• Express a gene at high level in a particular
  tissue
• Study the effects of high expression of gene on
  physiology
• eg myc and fos transgenics develop cancer
• high level expression may not reflect
  physiological role
• variable expression levels make different effects

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Gene knockouts

• Loss or mutation of a gene may be of great
  interest- how to study ?
• Embyonal Stem cells
• embryo derived, undifferentiated tumour cells
• can give rise to all tissue types
• when placed in an embryo, develop normally as
  part of the embryo

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ES Knockouts - 2
Inject into embryo
ES cell
Development
ES derived gonads
Breed
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Knockouts
Transfect DNA into ES cells
Neor
Thymidine kinase
Gene A
Random integration
Homologous recombination
Gene A has been replaced. Neor, but no TK
resistant to gancyclovir
Neor, but contains TK killed by gancyclovir
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Gene replacement

• Knockout the gene
• Mutate the gene
• What is its physiological role ?
• Particularly useful for
• Oncogenes what is their normal role ?
• Developmental genes- which genes are responsible
  for the various stages of development
• mouse model of human disease, eg cystic fibrosis

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Transgenesis

• available in animals and plants
• useful for protein production
• studying protein function
• gene regulation
• knockouts for studying physiological role of
  proteins
• plants- for improving plants