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.
Figure 22.13 from Snustad/Simmons Microinjection
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.