Biotech Applications: BIT 220 End of Chapter 22 (Snustad/Simmons)
Biotech Applications: BIT 220 End of Chapter 22 (Snustad/Simmons)
Biotech Applications: BIT 220 End of Chapter 22 (Snustad/Simmons)
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
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