Download Biotech Applications Transgenics, Nucleic acid therpeutics, Antibiotics

Biotech Applications
Nucleic acid therapeutics, Antibiotics, Transgenics
BIT 220
End of Chapter 22 (Snustad/Simmons)
Nucleic Acids as Therapeutic
Agents
• Many diseases (cancer, inflammatory
diseases) from overproduction of a protein
• Nucleotide sequences can help treat these
• Synthetic oligonucleotides:
– Single-stranded
– Several nt long
– Hybridize to mRNA – block or diminish
translation – called antisense oligonucleotide
Nucleic Acid Therapeutics
• Oligo that binds gene and blocks
transcription called an antigene
• Other ones can bind to transcription factor;
double-stranded oligonucleotides can bind
DNA binding proteins, stopping activation
of transcription
Antisense RNA
Antisense RNA
• Make transcripts in plasmid in wrong
orientation – can get gene sequence (but
doesn’t make correct protein)
• Examples where could be used:
– Insulin like growth factor 1; found in brain
cancer and prostate cancer
– Worked in mice – they developed smaller or
no tumors when cancerous prostate cells
were injected with antisense RNA (large
tumors in controls
Antisense Oligonucleotides
• Same idea as antisense RNA, but only
blocking a portion of the mRNA of the
molecule with a targeted synthesized oligo
• E.g. (really 15-25 nt. in length)
Sequence : 5’ AUCCUAGGA3’ (from mRNA)
Oligo:
UAGGAUCCU
Antisense Oligonucleotides
• May try different oligos that block different
portions of mRNA- see which one works
best at blocking translation
• No area better than others; have tried:
– 5’ and 3’ regions of molecule
– Places which were exon/intron boundaries
Antisense Oligonucleotides
• Other applications – restenosis – 40% of
patients get more arterial blockage after
angioplasty
• Oligos to cell cycle in rat arteries blocked
restenosis by about 90%
• In humans – antisense in phase I for
Crohn’s disease (inflammatory condition),
blocked intercellular adhesion molecule 1
• Few side affects
Antibiotics
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Amoxicillin
Penicillin
Erythromycin
Streptomycin
Tetracycline
Cefaclor
The list goes on……(about 12,000 kinds!)
Antibiotics
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Treat bacterial disease
Saved millions of lives
100,000 tons produced per year
$5 billion in sales
200 new ones per year are discovered,
only 1% are marketed
Antibiotic Biosynthesis Genes
• Biosynthetic pathways are complex
– 10-30 enzymatic steps
– Little/no information about genes or proteins
• Strategy to clone/isolate genes?
– Clone by Complementation
Molecular Biotech Role in
Antibiotics
• develop new, structurally unique
antibiotics with increased activities
– mixture of enzymatic pathways in same
organism (metabolic engineering)
• genetic manipulation to enhance yields
and thus lower cost of production
– Strategies to enhance production
Transformation of Streptomyces
Transgenic Animals
process of introducing foreign or
exogenous DNA into an animal’s
genome
Transgene
DNA introduced into:
Cows
Fish
Birds
Sheep
Mice
Goats
Transgenic Animals
• Single genes or gene clusters into the
chromosomal DNA of higher organisms
• Strategy:
– 1. cloned gene injected into nucleus of fertilized egg
– 2. inoculated eggs implanted into female
– 3. some offspring carry cloned gene all their cells
– 4. breed animals with germ-line integrated cloned
gene
Why transgenics?
• Improve genetic Features of domesticated
Animals
• Provide animal models for study of human
diseases
• Pharming
– using farm animals for production of human
pharmaceuticals
-mammary glands
– Study the genes regulation, development of animals
Definition
• Animal (or plant) whose genetic
composition is altered by the addition of an
exogenous, foreign gene is TRANSGENIC
• Methods:
– Embryonic stem cell
– Pronucleus method
Embryonic Stem Cell Method
• 1. Make your DNA
• Using recombinant DNA methods, the structural
gene you desire (e.g., the insulin gene)
• vector DNA to enable the molecules to be
inserted into host DNA molecules
• promoter and enhancer sequences to enable
the gene to be expressed by host cells
• 2. Transform ES cells in culture
• Expose the cultured cells to the DNA so that
some will incorporate it.
Embryonic Stem Cell Method
• 3. Select for successfully transformed cells. [
• 4. Inject these cells into the inner cell mass (ICM) of
mouse blastocysts.
• 5. Embryo transfer
• Prepare a pseudopregnant mouse (by mating a female
mouse with a vasectomized male). The stimulus of
mating elicits the hormonal changes needed to make her
uterus receptive.
• Transfer the embryos into her uterus.
• Hope that they implant successfully and develop into
healthy pups (no more than one-third will).
Embryonic Stem Cell Method
• 7. Establish a transgenic strain
• Mate two heterozygous mice and screen
their offspring for the 1:4 that will be
homozygous for the transgene.
• Mating these will found the transgenic
strain.
Pronucleus Method
• Prepare your DNA as in Method 1
• 2. Transform fertilized eggs
• Harvest freshly fertilized eggs before the sperm
head has become a pronucleus.
• Inject the male pronucleus with your DNA.
• When the pronuclei have fused to form the
diploid zygote nucleus, allow the zygote to divide
by mitosis to form a 2-cell embryo.
• 3. Implant the embryos in a pseudopregnant
foster mother and proceed as in Method 1.
Microinjection
Figure 22.13 from
Snustad/Simmons
Transgenic mouse- Human growth
hormone
Once Transgenics are made
Integration of DNA
G0 generation : Mosiacs
(G1 progeny): all cells contain transgene
1. Determine whether the progeny are
TRANSGENIC
piece of tail, PCR, Southern Blot
2. Mate this transgenic animal to determine
whether the transgene is in GERM line
3. Breed to get homogenous transgenic line
Transgenic animals - applications
• Model systems – biological basis of
human disease
• Better milk production from cows
• Bigger pigs (growth hormone)
• Chickens – low cholesterol eggs
• Fish – again, bigger – growth hormone
• Sheep – increased wool production
Transgenic Model for Alzheimer’s
• See handout
• Gene inserted has mutations of APP that
correlate with early onset of disease (<
age 50)
• Can help learn about progression of
disease, etc.