Animal biotech
What is pharmacokinetics?
the study of how drugs interact with tissues and how they are metabolized.
What can be examined in animal models?
the rate of absorption, specific chemical metabolism, and time required to excrete the substance.
What is the benefit of using mice
many genetic and physiologic similarities between mice and humans. Most often: pure-bred mice and rats (of which the genetics are known and controlled)
How is sperm-mediated transfer performed?
Use 'linker proteins' to attach DNA to sperm cells. When the sperm cell fertilizes an egg, it carries the valuable genes along with it.
What are alternatives to the use of animals
Use cell cultures and computer-generated models in initial testing (preliminary screen) to check toxicity of the substances.
Examples where animal research benefited human
Polo vaccine. Dialysis Cataract surgery techniques.
How can food be made more healthy?
transgenics. Tweaking the genes responsible for cholesterol production could result in healthier, lower-cholesterol eggs. Use transgenics to increase milk production, make milk richer in proteins and lower the fat content.
How is Nuclear Transfer to produce a cloned animal carried out?
usually begins with removal of the egg nucleus (possibly from an adult animal). If the process is successful, the desirable characteristics can be obtained with a high degree of certainty. Success is not always easy.
Give an example of the use of transgenic animal being used to produce pharmaceutical proteins?
A human gene called Atryn, responsible for an anticlotting agent in humans, is needed by patients with a hereditary deficiency to prevent thromboembolism before or after surgery or childbirth. The product was approved for use by the FDA in 2009, and although the market for patients with hereditary antithrombin deficiency treatable with ATryn is not large, it may prove to have other uses in the operating room. This transgenic system is less expensive using biorector animals and is under development for other proteins (like monoclonal antibodies) that are needed in large volumes.
How is retrovirus-mediated transgenics carried out.
Accomplished by infecting mouse embryos with retroviruses before the embryos are implanted. The retrovirus acts as a vector for the new DNA. This method is restricted in its applications because the size of the transgene (transferred genetic material) is limited and the virus's own genetic sequence can interfere with the process, making transgenics a rather hit-and-miss project.
What is selective improvements?
Allow animals to reach the market quickly by introducing gens that are responsible for faster growth rates or leaner growth patterns.
Give an example of animal testing benefiting animals and human
BRACA1 gene found in 65% of human breast tumors is similar to the BRCA1 gene in dogs. Clinical trials of cancer therapies on pet can pave the way for human trials.
What are examples of bioreactors
Biosteel, an extraordinary new product that may soon be used to strengthen bulletproof vests and suture silk in operating rooms. Spider genes for silk protein has been transferred into goats, to produce milk with silk protein. The silk protein is then purified from the goat's milk and woven into thread for fabrication into bullet-proof vests and other products.
What was the first knockout rat using the zinc-finger nuclease methodology
By the Sigma company in 2009. Knock-out rat models offer an alterntive that can reduce drug development costs by providing a more human like model while decreasing time to market. Zinc-finger nuclease-mediated gene targeting in the rat has opened up a whole new world of possibilities for the development of more predictive models of drug metabolism in humans.
How to create a clone from an adult?
Cells are collected from the donor animal and placed in a low-nutrient culture solution. The cells are alive but starved, so they stop division and switch off their active genes. The donor are then ready to be introduced into a recipient egg. The egg is prepared by enucleation. A pipette gently suctions out the DNA congregated in the nucleus of the egg. The researchers must then choose a method for delivering the desired genetic payload into the egg. After the DNA has been delivered to the egg, the new cell responds by behaving as if it were an embryonic cell rather than an adult cell. If all goes well, cell division occurs, just as it would in an ordinary fertilized egg. For the first few days, the embryo grows in an incubator. Then, when the new embryo is ready, it is transferred into a surrogate mother for gestation. The surrogate will give birth to a clone that is genetically identical to the genetic donor.
What is the future of cloning?
DNA from frozen cells has been used successfully in the cloning process, opening up new possibilities that will opend on cryogenetically preserved cells. Clones could be used to provide replacement body parts for their donor (reduce rejections) Xenotransplantation: use organs from other species. used in medical research, in which their identical genetics makes it easier to sort out the results of treatments without the confounding factor of different genetic predispositions. Provide unique window on the cellular and molecular secrets of development, aging, and diseases. Clone endangered species to sustain breeding populations.
What are the limitations to cloning?
Donor cell must come from a living organism. Though the clones are exact genetic duplicates, they are not exactly identical to the original. Animals are shaped by their experiences and environments as well as their genes. Present success rate for cloning is actually quite low. Many clones are born with defects. The mortality rate among the surrogate mothers is much higher than among normal mothers. Clones may become old before their time due to telomeres shortening.
How is embryonic stem cell method carried out?
ES cells are collected from the inner cell mass of blastocytes. They are then mixed with DNA, which has usually been created using recombinant DNA methods. Some of the ES cells will absorb the DNA and be transformed by the new genetic material. Those transformed ES cells will then be injected into the inner cell mass of a host blastocyst.
What is an efficient method of producing biotech products in animals?
Eggs. Poultry have an efficient reproductive system, it is easy to generate thousands of eggs from a small flock in a relatively short time. An average hen produces 250 eggs per year. With each egg white containing about 3.5g of proteins, replacement of even a small fraction with recombinant protein creates highly productive 'hen bioreactor'. Some medical products such as lysozyme and attenuated virus vaccines are already derived from eggs.
What is the first successful experiment on cloning?
Embryo twinning: produce two perfectly healthy calves that were essentially twins.
What is the limitation with embryo twinning
Even though the end result is identical twins, the exact nature of those twins is the result of mixing the genetic material from two parents. You may end up with animals that have the desired characteristics, but you have to wait until they are grown to find out.
What are the limitatiosn with cell cultures?
Give clues about which compounds might make the best drugs, but cannot provide information about the potential impacts on an entire living organism.
When are mice, rats and fish not the best test animals?
HIV/AIDS research conducted on monkeys and chimpanzees because they share human's vulnerability to the virus. use dogs to study lung and cardiovascular diseases.
Give an example of the use of transgenics in animals
Herman, a transgenic bull (produced by transgenics and nuclear transfer), carries the human gene for lactoferrin. This gene is responsible for the higher iron content (essential to the healthy development of infants) found in human milk. Herman's offspring also carry the gene for lactoferrin. Consequently, in much the same way that human polyclonal antibodies can be produced in the plasma of cattle. Herman's daughters may be the first of many cows able to produce milk that is better suited for consumption by human children.
What is the difference betwen reaction in gene knock-out and knock-in mouse.
In a knock out mouse, the drug would have no or minimal effects because the gene product was not present, but it would have effects in the presence of the human gene in the knock-in mouse. If effects are seen in the knockout animal they are off target and can represent potential side effects of the candidate drug (saving human suffering without having to test humans).
What are the advantages of transgenic food?
Increase agricultural production. Food could be made more healthy. Reduce diseases in animals raised for food. Reduce human food poisoning by transfering antimicrobial genes to farm animals, fewer antibiotics used in agriculture. Create animals that will better serve hungry and poor populations around the world.
How to deliver the desired genetic payload into the egg by the Honolulu technique?
Injecting the nucleus of the donor cell directly into the middle of the enucleated egg cell.
What is an alternative to homologous recombination?
Interference in RNA is also capable of gene silencing and being utilized as an alternative to homologous recombination in many applications.
How is pronuclear microinjection carried out?
Introduce the transgene DNA at the earliest possible stage of development of the zygote (fertilized egg). When the sperm and egg cells join, the DNA is injected directly into the nucleus of either the sperm or the egg. BEcause the new DNA is injected directly, no vector is required; therefore there is no external genetic sequence to muddle the process.
How has research at veterinary clinics benefit human and animals
Led to new cancer treatments for pets. animals and human cancer are strikingly similar, information gleaned from one species might be used to treat the other.
What animals are used in research
Mice vertebrates: zebrafish invertebrates: fruit flies Worms
How is knockout mice created
Modified DNA added to ES cells where it recombines with the existing gene on a chromosome, essentially deleting or replacing part of the genetic instructions in the cell. The modified ES cells are then introduced into a normal embryo, and the embryo is implanted in an incubator mother. The offspring of the mouse pup is a chimera.
Why can't you just test on cells in vitro?
New drugs and medical procedures have effects beyond single cells in tissues and organs and must be tested in animals to determine the impact of the treatment on an entire organism. e.g. a drug may be a powerful agent able to kill cancer cells, but it may also be destructive to unrelated organs.
What are the two methods to deliver the desired genetic payload into the egg?
Nuclear Transfer Honolulu technique
How to create MAbs that does not activate the human immune system to rejection
Panitumab, produced from transgenic mice with knock-in human immune genes. The process involved inactivating the mouse antibody machinery adn introducing the human equivalent of these antibody-producing genes by homologous recombination into the inactivated (deleted) regions. The transgenic mice produce a fully human antibody against epidermal growth factor receptor, which can be purified and does not produce the HAMA resposne.
What is the limitation in the therapeutic use of antibodies
Problem of producing a specific antibody in large quantities.
Give an example where experiments using animal subjects help prevent unexpected disasters?
Propecia (finasteride), a drug for hair growth carries a clear warning that pregnant women should not handle broken or crushed pills. That warning and a special protective coating on the pills themselves are the direct result of information collected during testing on animal subjects. The tests revealed serious birth defects (malformations of the reproductive organs) in male offspring born to animal mothers given large oral doses of the drug. Although merely handling the pill is unlikely to result in similar defects in human infants, the warnings were put in place before the drug was marketed.
What are the Three Rs of animal research
Reduce the number of higher species (cats, dogs, primates) that are used. Replace animals with alternative models whenever possible. Refine tests and experiments to ensure the most humane conditions possible
Give an example of gene knock-in
Researchers at the Oregon Regional Primate Research Center at the Oregon Health Sciences University in Portland have introduced the gene for green fluorescent protein (GFP) into a rhesus monkey named ANDi ('inserted DNA' spelled backwards). The GFP gene, which had its origin in a jellyfish, does not cause ANDi to glow in the dark. It looks exactly like a regular rhesus moneky. It takes a special microscope to see the glow in ANDi's cells. The demonstration that a gene can be inserted into monkeys was a major accomplishment to prove that the method can work in higher animals, even if it never results in a significant medical application.
What must researchers do to receive funding for research from the National Institutes of Heath (NIH), the FDA, or the Centers for Disease Control and Prevention (CDCP)?
Researchers must follow the standards of care set out in The Guide for the Care and Use of Laboratory Animals, formulated by the National Academy of Sciences.
What are the transgenic techniques
Retrovirus-mediated transgenics. Pronuclear microinjection. Embryonic stem cell method. Sperm-mediated transfer. Gene guns.
What is gene knockout
The DNA is prepared using recombinant DNA techniques either to clip away a specific portion, leaving a deletion in the genetic code, or to replace one portion of the DNA with a snippet of DNA that has no function. The resulting action would be different from the action that the original instruction was intended to produce. Deleting or replacing a bit of DNA can result in a change in the traits expressed, including termination of teh expression of vital proteins.
What are the regulations of animal research
The Federal Animal Welfare Act. Government agencies regularly monitor the conditions in the laboritories. An oversight committee with the host institution reviews the research while it is in progress to make certain that institutional and federal standards are being upheld.
Why use transgenic goat to link ATryn to mammary-specific promotors
The company that developed the transgenic goat (GTC) linked ATryn to mammary-specific promotors so that it would be expressed in milk. The transgenic method is faster, more reliable, and cheaper than other more complex ways of synthesizing pharmaceutical proteins.
How does an animal bioreactor work?
The gene for a desired protein is introduced via transgenics to the target cell. Use cloning techniques, the cell is raised to become an adult animal. That adult may produce milk or eggs that are rich in the desired protein, thanks to the introduced gene.
How animal research has directly benefit animal health?
Treat heartworm, arthritis, parasites, allergies, heart disease. Vaccines to prevent rabies and feline HIV.
What are the advantages of using transgenic animal bioreactors for the production of complex therapeutic proteins?
provides lower production costs, higher production capacities, and safer pathogen-free products.
What does the FDA require manufacturers from phase testing (regulations in animal research) ?
conduct a statistically significant number of trials on cell cultures as well as trials in living animals and human research participants before such products can be made available to the general public. Have to pass each phase before moving to the next phase.
What motivates researchers to seek funds from NIH, FDA, or CDCP
cost of research, the availability of grant monies from those agencies can be essential.
How to choose animals for testing?
determined by their genetic similarity to humans or potential to mimic conditions in humans.
Why are two or more species used in the tests
different effects may be revealed in different animals. Toxicity and unexpected nontarget effects can often be detected by using more than one species with genes homologous to those of humans.
What is the purpose of gene knockouts
disrupt a specific gene. An active gene is replaced with DNA that has no functional information. When the gene is knocked out of place by the useless DNA, the trait controlled by the gene is eliminated from the animal.
Why research in transgenics often use goats?
goats reproduce more quickly and are cheaper to raise than cattle. They also produce abundant milk.
What is the purpose of traditional cross-breeding techniques?
increase growth rate increase production levels decrease cost.
Why has the number of animal testing declined in the past 20 years?
increased use and availability of alternative methods of testing.
Advantages of cell cultures and computer-generated models?
less expensive than research animals. save time.
What are the limitations with computer models?
limited by their programming. They can provide clues, but they cannot provide final answers. Unless we already know the impact on a living system, we cannot create a computer program that can simulate that system. Cannot react unless we have programmed them to do so.
What does safety testing involve
phase testing: rigorous scientific methodology
What is the advantage of embryo twinning?
produce many cattle with the same strong blond line. Common practice in the cattle industry today.
What are the problems with producing MAbs using mouse hybridoma cells
provoke immune response in human patients - patients quickly eliminated mouse MAbs, so the therapeutic effects were short-lived (Human antimouse antibody HAMA response): Solution is to make MAbs more human. Remove mouse antigens or create chimeric cells is costly and time-consuming.
What is homologous recombination
recombine ES cells with modified DNA with existing gene on a chromosome, to delete or replace part of the genetic instruction in the cell.
What are some motivations for some of the current research?
reduction int he time required for production would significantly save money for producers, which could then be passed on to consumers.
How does the Federal Animal Welfare Act regulate animal research
sets specific standards concerning the housing, feeding, cleanliness, and medical care of research animals. Before a study using animals can begin, researchers are required to prove the need to employ animals. They must also select the most appropriate species and devise a plan for using as few animals as possible.
What are the benefits of using zebrafish?
small: possible to house large numbers of animals in small spaces. Spawning is virtually continuous, with only about 3 months between generations. Zebrafish eggs complete embryogenesis in about 120 hours. Because the fish grow so rapidly, scientists can test for toxicity or adverse effects in about 5 days. The rapid growth of an visible embryo inside the zebrafish's egg makes studies efficient. The eggs lend themselves readily to gene transfer, and the embryos are transparent, making it possible to study cell division under the microscope in its first hours.
What must new drugs, medical procedures, and even cosmetic products must past before they can be marketed.
the US Food and Drug Administration (FDA) requires them to pass safety tests before they can be marketed.
Why are rats superior to mice for many early drug toxicity tests?
their physiology more human like responses to drugs. The relatively large size of rats facilitates surgical and physiological experimentation. This is particularly important in studies that require dissection or isolation of specific cell types and organ structures, as is the case with pharmacokinetics. Rats are the preferred model organism for determining drug metabolism and early toxicology profiles.
What is the purpose of gene transfer?
to improve the productivity of livestock.
Why are computer-generated models used?
to simulate specific molecular and chemical structures and their interactions. They are becoming increasingly sophisticated. Excellent for processing data and helping us see the patterns that emerge.
What are the most attractive alternative for replacement therapy in genetic disorders such as hemophilia?
transgenic-derived therapeutic proteins. Clotting protein antithrombin III is being produced in transgenic goats and being tested for FDA approval.
