Outcome 5 Flashcards
What is a model organsim
A non-human species studied to understrand a fundamental biological phenomena or provide insights into other organisms
Which model organism is useful for studying genetic inheritance
Pea Plant for Mendelian inheritance or fruit fly
Which model organism is useful for studying cellular processes
Yeast
Which animal model is particularly useful for studying embryology
Zebrafish
Give three traits/properties of a good model organism.
Quick generation time, easy to grow.
Similarity in physiology, genetics, anatomy when compared to humans.
Name the most commonly used mammals for experimental studies of human disease?
Rodents - house mouse and brown rat
What is meant by the 3 Rs? Describe them.
Reduction, refinement, replacement.
Reduction - reduce number of animals needed to conduct experiment with improved experimental design. Share information with other researchers.
Refinement - reduce suffering by using less invasive techniques. Anaesthitising animals when possible. Providing better living conditions and handling.
Replacement - Alternatives to model organisms used when possible such as cell cultures, human volunteers.
What are some limitations of using an animal model
Some results obtained on animals are not confirmed in further human studies due to differences in physiology, genetics or anatomy.
Genetic and physiological variations within a species can affect the results of an experiment.
What is meant by an inbred strain? What is the advantage of using inbred strains (e.g.
of mice) for experiments?
Laboratory mice have been developed as inbred strains which have highly homogeneous genetic composition to increase the reproducibility of results and the statistical power of experiments.
Define the term transgene.
Transgene refers to foreign DNA that is transferred into an animal via DNA recombinant technology and is subsequently transmitted through the germ line so that every cell in the body contains the modified genetic material.
Name the three principal methods for the creation of transgenic animals?
DNA microinjection
Embryonic stem cell mediated gene transfer
Retrovirus mediated gene transfer
Describe the 1st step in the process for generating a transgenic mouse using DNA microinjection
Make the transgene - using recombinant DNA methods, the desired gene, vector DNA, and promoter and regulatory sequences
Describe the 2nd step in the process for generating a transgenic mouse using DNA microinjection
In vitro fertilisation - female mouse is superovulated and eggs are fertilised in vitro
Describe the 3rd step in the process for generating a transgenic mouse using DNA microinjection
Transform fertilised eggs - male pronucleus is injected with the recombinant DNA. Once the pronuclei have fused to form a diploid zygote nucleus, the zygote divides to form a 2 cell embryo
Describe the 4th step in the process for generating a transgenic mouse using DNA microinjection
Implant embryos in pseudopregnant mother - The embryo is transferred into the oviduct of the recipient and the foster mother is induced to act as a recipient by mating with a vasectomised male
Describe the 5th step in the process for generating a transgenic mouse using DNA microinjection
Test offspring for transgene - remove tissue and test for transgene via PCR. Heterozygous offspring are mated with homozygous transgenic strain to produce homozygous transgenic strain.
What is meant by the term chimera?
An organism composed of cells from more than one distinct genotytpe
What is the first step in the process of embryonic stem cell mediated gene transfer
Embryonic ctem cells are harvested from the inner cell mass of the animals blastocyst
What is the 2ndt step in the process of embryonic stem cell mediated gene transfer
Make the transgene - recombinant DNA methods used to build a DNA molecule containing the desired gene, vector DNA, promoter and regulatory sequences
What is the 3rd step in the process of embryonic stem cell mediated gene transfer
Transform the embryonic stem cells - mix the cells with the recombinant DNA, and perform electroporation. Select for transformed cells
What is the 4th step in the process of embryonic stem cell mediated gene transfer
Inject the transformed cells into the inner cell mass of the organisms blastocyst
What is the 5th step in the process of embryonic stem cell mediated gene transfer
Implant the embryos into the pseudopregnant mothers oviduct, induce foster mother to act as recipient by mating with vasectomised male
What is the 6th step in the process of embryonic stem cell mediated gene transfer
Test offspring for transgene, the offspring are chimeric
What is the 7th step in the process of embryonic stem cell mediated gene transfer
Establish a transgenic strain - mate chimera with an albino mouse. Offspring tested to confirm presence of transgene via PCR. If transgene is present then the offspring are heterozygous. MAte heterozygous transgene containing offspring together to establish homozygous transgenic strain
What is the major advantage of using retroviruses to mediate gene transfer?
The retrovirus acts as a vector, retroviruses are well evolved to infect host cells
Describe the process of retrovirus mediated gene transfer
The retrovirus is used as a vector for the gene of interest, the retrovirus infects the germline (random insertion). Offspring are often chimeric because not all cells are infected. Germ line chimeras are then inbred for 10- 20 generations until homozygous transgenic animals are produced where the transgene is present in every cell
Define random gene insertion
The gene is inserted into the animal at a random gene locus and a random number of times (Retrovirus mediated)(microinjected)
Define targeted gene insertion
The gene is transfered into the genome at a specific locus (ES mediated)
In the vector, the gene of interest (and
promoters/enhancers/selection marker) must be
flanked by sequences that are homologous to the
targeted gene/genome sequence
What are knockout mice
When targeted gene is replaced with a nonfunctional gene, no/less protein expression is caused and loss of function. Used to study the function of genes and their role in disease
What are knock-in mice
Knock-in mice are produced by inserting a transgene into an exact location where it is over expressed.
Outline how animal models can be used to investigate genetic disease with regards to disease mechanism
As animals often do not suffer from the same diseases as humans, the generation of transgenic animals (usually transgenic mice) is often necessary to investigate genetic disease. This is useful as model animals can have genomes similar enough to humans to use them for research into a genetic disease which is linked to a gene mutation (sickle cell anemia, cancers) The transgenic animal can be used to study the mechanism of the disease as well as how the disease progresses. Expression of the gene can be studied to learn in what cells and when it is expressed. The effect of drugs or new trreatments can be studied using the model animal, with a view to confirming finding with later human clinical trials.
Outline how animal models can be used to investigate genetic disease with regards to analysing gene expression
The function of many human genes are still poorly understood. By using knockout mice, the effects of selectively removing a gene (and therefore stopping corresponding protein production) can be observed. Humans and mice share many genes and the results may therefore be relevant to humans. Knockout mice have been used to study different types of cancer and Parkinsons disease. The p53 gene supresses tumour development by arresting cell development. By selectively removing this gene in knockout mice, the role that mutations to the p53 gene in humans (such as increased risk of bone cancer) can be better understood
