Biotech Applications Transgenics, Nucleic acid therpeutics, Antibiotics

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Dec 5, 2012 (4 years and 11 months ago)

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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


Amoxicillin


Penicillin


Erythromycin


Streptomycin


Tetracycline


Cefaclor


The list goes on……(about 12,000 kinds!)

Antibiotics


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.