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Transgenic mice

This document provides an overview of transgenic mice, which are genetically modified organisms containing foreign genes to study various human diseases and traits. It discusses the history, methods of creation, and applications of these mice, particularly in modeling diseases and drug testing. Additionally, it highlights the significance of knockout mice in understanding gene functions and their role in genetics research.

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transgenic-mice By KAUSHAL KUMAR SAHU Assistant Professor (Ad Hoc) Department of Biotechnology Govt. Digvijay Autonomous P. G. College Raj-Nandgaon ( C. G. )
Synopsis • Introduction • Definition • History • Why are the transgenic animals being produced • Transgenic mice • Mice: as model organism • Methods of creation of transgenic mice • knock-out mice • Application of transgenic mice • Conclusion • References
Introduction:- transgenic animal • A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome. The foreign gene is constructed using recombinant DNA technology. In addition to the gene itself, the DNA usually includes other sequences to enable it • To be incorporated into the DNA of the host and • To be expressed correctly by the cells of the host. • Transgenesis refers to the phenomenon of introduction of exogenous DNA into the genome to create and maintained a stable heritable character. • The majority of transgenic animals produced so far are mice, the animal that pioneered the technology. The first successful transgenic animal was a mouse. A few years later, it was followed by rabbits, pigs, sheep, and cattle.
transgenic mice • A transgenic mice contains additional, artificially- introduced genetic material in their genome. The extra genetic material is often described as foreign DNA, but it can come from any source, including another mouse. • To get the same foreign DNA sequence into every cell of the mouse, it is necessary to introduce the DNA into cells of the very early mouse embryo that will contribute to the germ line (the cells that produce eggs or sperm).
Definition:- transgenic mice • A transgenic mice is one that carries a foreign gene that has been deliberately inserted into its genome. The foreign gene is constructed using recombinant DNA methodology.
History • In 1972: creation of the first recombinant DNA molecules by Paul Berg • In 1973: the first GMO was created by Stanley N. Cohen and Herbert Boyer, demonstrating the creation of a functional organism that combined and replicated genetic information from different species. • In 1974: the first transgenic animals were mice created by Rudolf Jaenisch. • Jaenisch successfully managed to insert foreign DNA into the early-stage mouse embryos, the resulting mice carried the modified gene in all their tissues. Subsequent experiments, injecting leukemia genes to early mouse embryos using a retrovirus vector, proved the genes integrated not only to the mice themselves, but also to their progeny.
Why are these animals being produced? • The two most common reasons are: • Transgenic animals are useful as disease models and producers of substances for human welfare. • Some transgenic animals are produced for specific economic traits. For example, transgenic cattle were created to produce milk containing particular human proteins, which may help in the treatment of human emphysema. • Other transgenic animals are produced as disease models. For example, genetically engineered mouse, called Onco Mouse carrying a gene that promotes the development of various human cancers.
Mouse: as model organism • Mice is an excellent genetic model of vertebrate development • The sequence and analysis of a mouse strain in December 2002 the mouse became the animal model of choice for most laboratory experiments. • Mouse and human development are very similar. • The mouse makes an excellent model for human disease because the organization of their DNA and way their genes are expressed is very similar to humans. • Their reproductive and nervous systems are like those of humans, and they suffer from many of the same diseases such as cancer, diabetes and even anxiety. • Manipulating their genes can lead them to develop other diseases that do not naturally affect them, and as a result research on mice has helped understanding of both human physiology and the causes of disease. Fig:- A model for human genetic diseases: when certain genes are knocked out in mice, the resulting phenotype often resembles a human inherited disease (i.e. loss of steel factor receptor in mice leads to a phenotype similar to piebaldism in humans). These mice are an excellent model to study how gene therapy can be used to restore the normal gene and correct the disease. White spotting : Piebaldism Kit gene
Model organisms: mice • Advantages: • vertebrates! • Large Genome = 3 Gb • mice are ~ 3 inches long, can keep many mice in a room. • generation time is ~ 3 months, so genetics can be done • history - scientists have worked with mice for 100 years • genetic tools - can introduce extra genes or remove a specific gene, then study the effect on development • Disadvantages: • development inside the mother, hard to see. • Expensive!
Methods for making transgenic mice • There are three basic methods of producing transgenic mice: DNA microinjection Embryonic stem cell-mediated gene transfer Retrovirus-mediated gene transfer • The insertion of a foreign gene (transgene) into an animal is successful only if the gene is inherited by offspring. • The success rate for transgenesis is very low and successful transgenic animals need to be cloned or mated.
Method:-DNA microinjection • This method involves the direct microinjection of a chosen gene construct (a single gene or a combination of genes) from another member of the same species or from a different species, into the pronucleus of a fertilized ovum. • It is one of the first methods that proved to be effective in mammals (Gordon and Ruddle, 1981). • The introduced DNA may lead to the  over- or under-expression of certain genes or  to the expression of genes entirely new to the animal species. • The insertion of DNA is, however, a random process, and there is a high probability that the introduced gene will not insert itself into a site on the host DNA that will permit its expression. • The manipulated fertilized ovum is transferred into the oviduct of a recipient female, or foster mother that has been induced to act as a recipient by mating with a vasectomized male.
1 FIGURE 1 Production of transgenic mice by pro-nuclear microinjection. Female mice are superovulated and mated, and a pronucleus of the fertilized egg is microinjected with DNA (top right). Surviving zygotes are reimplanted (center), and newborn pups are tested by Southern blotting for incorporation of new genes (bottom). Coat color markers are use to identify microinjected embryos.
Method:-DNA microinjection • A major advantage of this method is its applicability to a wide variety of species. • Most commonly used method • Only 5% or less of the treated eggs become transgenic progeny • Need to check mouse pups for DNA ( by PCR or Southerns), RNA ( by Northerns or RT-PCR), and protein ( by Western or by some specifis assay method) • Expression will vary in transgenic offspring: due to position effect and copy number
Method:- Embryonic stem cell-mediated gene transfer • This method involves prior insertion of the desired DNA sequence by homologous recombination into an in vitro culture of embryonic stem (ES) cells. • Stem cells are undifferentiated cells that have the potential to differentiate into any type of cell (somatic and germ cells) and therefore to give rise to a complete organism. • These cells are then incorporated into an embryo at the blastocyst stage of development. • The result is a chimeric animal. ES cell-mediated gene transfer is the method of choice for gene inactivation, the so-called knock-out method.
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Method:- Retrovirus-mediated gene transfer • To increase the probability of expression, gene transfer is mediated by means of a carrier or vector, generally a virus or a plasmid. • Retroviruses are commonly used as vectors to transfer genetic material into the cell, taking advantage of their ability to infect host cells in this way. • Offspring derived from this method are chimeric, i.e., not all cells carry the retrovirus. • Transmission of the transgene is possible only if the retrovirus integrates into some of the germ cells.
Example of Transgenic Strains • Onco mice, have an inactivated oncogene, and are predisposed to developing cancer. These mice have been vital to the understanding of many cancers and the development of technologies to treat them. • Super mice, (R. L. Brinster and R. E. Hammer) The giant mouse developed from a fertilized egg transformed with a recombinant DNA. The levels of growth hormone in the serum of some of the transgenic mice were several hundred times higher than in control mice. • Doogie mice show improved memory and capacity for learning. These mice have enhanced function at NMDA receptors, which are needed for the brain to store new information etc.
Example of Transgenic Strains • Cancer-Resistant Mouse • In their paper published in Cancer Research in 2007, University of Kentucky scientists revealed a startling finding: a mouse that was resistant to spontaneous and artificially induced tumors. Its inability to get cancer is due to the insertion of a gene that codes for a protein called Par-4. This protein specifically kills cancer cells without affecting normal cells, which according to researcher Vivek M. Rangnekar makes it "a tumor suppressor that would be ideal for therapeutic intervention strategies.” Mighty Mice The mice in the laboratory of Dr. Richard Hanson at Case Western Reserve University have earned their nickname: they can run for 25 times as long as normal mice at the same speed, and they eat more and live longer. Bred as a tool to investigate the metabolic enzyme PEPCK-C, which plays a role in the generation of glucose, the mice are genetically altered to over-produce this enzyme.
Knock-out Mice • The more recent development of knock-out strains of mice during the 1980’s was a major advance for genetics. • This allows researchers to determine the exact function of a particular gene, and these GM mice have provided excellent models of many human diseases. • The sequencing and analysis of the mouse genome has allowed many genes to be targeted and studied using this technology. • The creators of the first knockout mice were awarded the 2007 Nobel Prize in medicine. • The technique which led to the creation of knockout mice was developed in bacteria by Joshua Lederburg, who received the Nobel Prize for his discovery in 1958. • A knockout mouse is a genetically engineered mouse in which researchers have inactivated, or "knocked out," an existing gene by replacing it or disrupting it with an artificial piece of DNA. The loss of gene activity often causes changes in a mouse's phenotype, which includes appearance, behavior and other observable physical and biochemical characteristics. • Knockout mice are important animal models for studying the role of genes which have been sequenced but whose functions have not been determined. By causing a specific gene to be inactive in the mouse, and observing any differences from normal behaviour or physiology, researchers can infer its probable function.
How does it work? • Knockout mice are produced by a technique called gene targeting. This is the replacement of one gene sequence, the sequence resident in the mouse genome, with a related sequence that has been modified in the laboratory to contain a mutation. • The replacement occurs by a process called homologous recombination, where two very similar DNA sequences line up next to each other and exchange parts. • Gene targeting is carried out in mouse embryonic stem cell (ES Cell). These cells are derived from a very early (usually male) mouse embryo and can therefore differentiate into all types of cell when introduced into another embryo. The aim is to get the modified ES cells to contribute to the germ line, which gives rise to sperm. Some sperm are produced that carry the desired mutation, and if these fertilise a normal egg, mice develop with one copy of the mutated gene in every cell.
3 Where •A = original gene •A*= replacement gene •neor, a gene that encodes an enzyme that inactivates the antibiotic neomycin and its relatives, like the drug G418, which is lethal to mammalian cells; tk, a gene that encodes thymidine kinase, an enzyme that phosphorylates the nucleoside analog ganciclovir. DNA polymerase fails to discriminate against the resulting nucleotide and inserts this nonfunctional nucleotide into freshly-replicating DNA. So ganciclovir kills cells that contain the tk gene Fig:- gene targeting
o • interbreeding such mice will produce some homozygous individuals in the next generation – mice inheriting the mutation from both parents and therefore carrying two copies of mutant gene. These are knockout mice.
How is it used? • The phenotype of a knockout mouse provides important clues about the genes normal role. • One major application of this technology is the modeling of human diseases caused by loss of gene function. • Examples include cystic fibrosis, beta-thalassaemia and various forms of cancer. • Such models are useful because they can be used to investigate the biochemical and physiological aspects of the disease and for the development and testing of drugs.
Examples of knockout Mice • There are many examples of knockout mice, as this technique has been used to study all aspects of physiology and to create models for many human diseases. • Fat mice, due to a carboxypeptidase E-deficiency. • Strong mice, with a disabled myostatin gene. • Cold-tolerant mice, lacking sodium channel which causes pain when exposed to cold.
Application of Transgenic Mice • Genetically modified mice are used extensively in research as models of human disease. • They have been used to study and model obesity, heart disease, diabetes, arthritis, substance abuse, anxiety, aging and parkison disease. • In toxicology: as responsive test animals (detection of toxicants) • In mammalian developmental genetics. • In molecular biology, the analysis of the regulation of gene expression makes use of the evaluation of a specific genetic change at the level of the whole animal.
Application of Transgenic Mice • In the pharmaceutical industry, targeted production of pharmaceutical proteins, drug production and product efficacy testing. • The disease symptoms and potential drugs or treatments can be tested against these mouse models. • The most common type is the knockout mouse, where the activity of a single (or in some cases multiple) genes are removed. • Transgenic mice generated to carry cloned onco genes and knockout mice lacking tumor suppressing genes have provided good models for human cancer. • Hundreds of these onco mice have been developed covering a wide range of cancers affecting most organs of the body and they are being refined to become more representative of human cancer.
Conclusion • A transgenic mouse contains additional, artificially- introduced genetic material in every cell. Transgenic mice are widely used in researching the characteristics of exogenous genes • A transgenic mouse a very useful system for studying mammalian gene function and regulation because analysis is carried out on the whole organism. • Transgenic mice are also used to model human diseases that involve the over expression or misexpression of a particular protein.
References:- • Books: • Molecular biology & recombinant DNA technology: glick & Pasternak • Biotechnology: U. Satyanarayan • Biotechnology: B. D. Singh • Websites: • www.wikipedia.com • www.kbiotech.com • www.sciencetechno.com