Transgenic AnimalsA genetically modified organism (GMO) is an organism whose genetic material has been altered using techniques in genetics, generally known as recombinant DNA technology. Recombinant DNA technology is the ability to combine DNA molecules from different sources into the one molecule. Thus, the abilities or the phenotype of the organism, or the proteins it produces, can be altered through the modification of its genes. Transgenics are genetically modified organisms with DNA from another source inserted into their genome. A large number of transgenic animals have been created Production of Transgenic Animals Transgenic animals can be produced by three different techniques: Method 1. Microinjection of DNA into the pronucleus of zygotes Method 2. Method 3. Use of engineered retroviruses The procedure is done with the help of a complete microinjection set-containing of a . Injection of embryonic stem cells into blastocysts. after the sperm enters the ovum. but before they fuse.A pronucleus (plural: pronuclei) is the nucleus of a sperm or an egg cell during the process of fertilization. . The foreign DNA must be integrated into the genome prior to the doubling of the genetic material that precedes the first cleavage in order for the animal to be born with a copy of this new information in every cell. The foreign DNA may be injected into either pronuclei with no difference in results. the male and the female pronuclei can still be seen individually under a normal light microscope and they have not fused yet into a so called zygote. the DNA is typically injected into the male pronucleus because it is slightly larger and closer to the oocyte surface. These oocytes are subsequently transferred into the uterus of pseudo-pregnant recipient animals. The offspring is .Method 1: Microinjection of DNA into the pronucleus The pronuclear microinjection method of producing transgenic animals is based on the introduction of linear DNA sequences into the chromosomes of fertilized eggs. For several hours following the entry of the sperm into the oocyte. however. the transgene is injected into the male pronucleus. • The offspring are screened for stable integration of the transgene. • The “foster mother” mouse has been treated with hormones so that she accepts the embryo and carries on with the pregnancy. • Harvested eggs and sperm are fertilized. and before the pronuclei fuse.Creation of Transgenic Animals by Nuclear Injection • In vitro fertilization is used to start a transgenic animal. Gene injected into the male pronuclei . • The embryo continues to divide in culture and is then implanted into a mouse. . site-specific recombination. ES cells are used for more precise modifications of the mouse genome. cloning and screening methods make it possible to detect ES cell clones that demonstrate the desired. These cells are pluripotent. selection. and conditional mutant mice can be produced with this method. After microinjection of the genetically modified ES cells into blastocyst-stage embryos. Knock-out. This technique makes it possible to insert as well as remove or modify DNA sequences. which means that they can develop into almost any type of tissue. The first step is the removal of ES cells from a blastocyst. After transfection of the ES cells.Method 2: Injection of ES cells into blastocyst-stage embryos Embryonic stem cells (ES cells) are derived from the inner cell mass of blastocysts (early embryos). the ES cells divide and become . which integrates by homologous recombination. Then the stem cells are injected into an early male embryo (blastocyst) of mouse and the embryo is put into a pseudopregnant female mouse.To create a transgenic animal with embryonic stem cells. the transgene must first be inserted into the embryonic stem cells. The stem cells are transformed with the transgene. The offspring are chimeras because . because insertion of the virus does not occur exactly when the nuclei fuse. •Retroviruses can carry only limited amounts of DNA. Hence.Method 3: Use of engineered retroviruses. Disadvantages •Virus DNA is introduced along with the transgene. •Founder animals made using retroviruses are always chimeras. retrovirus vectors may introduce transgenes. including embryonic stem cells. . Retroviruses can infect the cells of early embryos. Partially transgenic animals (that have some sectors or tissues altered) are useful because the transgenic tissues may be compared with normal tissue within the same animal. . but it was present at such low levels that it was not profitable to extract them.first animals were mice created by Rudolf Jaenisch in 1974. A human gene (responsible for production of lactoferrine) was inserted into bull’s genetic code while in an early embryonic stage in 1990. Research into animal and human disease Usetransgenic of animals as bioreactors The3. which can be used as medicine. There has also been the genetically manipulated bull.Goals of creation transgenic animal 1. milk from his female descendants contained the human protein lactoferrine. Improve livestock animals 2. the resulting mice carried the modified gene in all their tissues. Jaenish successfully managed to insert foreign DNA into the early-stage mouse embryos. As a result. •Instead of being made in the pituitary gland (the normal site for growth hormone) the rat somatotropin was manufactured in liver Human somatotropin has also been expressed in mice . although not as large as rats. The transgenic mice were larger (about twice normal size). Somatotropin gene of rat was cloned and inserted into fertilized mouse eggs.Large Transgenic Mice illustrate transgenic technology Creation of larger mice by inserting the rat gene for growth hormone (Somatotropin) Somatotropin consists of a single polypeptide encoded by a single gene. metallothionein . •The gene was under the control of the promoter from an unrelated mouse gene. which is normally expressed in the liver. Large Transgenic Mice . a protein that slows muscle growth. This is twice as far as a normal mouse can last. The cloned human version of the NR2E1 gene has been shown to cure the defects in brain development and restore normal behavior to fierce mice.Trendy Transgenic Mice Marathon Mouse Can run about 1800 meters—more than a mile—before exhaustion. Smart Mouse Having improved learning and memory. Marathon mouse has enhanced PPAR-delta—a regulator of several genes involved in burning fat and in muscle development. Fierce Mouse Shows abnormal aggression. Mighty Mouse Lack myostatin. Has extra copies of the NR2B gene encoding the NMDA . Both copies of the NR2E1 gene deleted. The result is colossal muscle development. The cloned genes are placed under the control of a regulatory region that will allow gene expression only in the mammary gland. transgenic goats producing rTPA (recombinant tissue plasminogen activator) . For small-scale production. transgenic goats are often used. E.Recombinant protein production using transgenic livestock Production of heterologous proteins Recombinant proteins can be produced in the milk from transgenic cows or other farm animals.g. homozygous . The incoming DNA. Founder mice are obtained that have one copy of the disrupted gene. replaces the original & functional copy of the gene by homologous recombination. mostly mice. are of great value in the genetic analysis of inherited diseases and cancer.” the gene of interest and then ask what defect this causes. mice with both copies disrupted are obtained. The general approach is to inactivate. carrying the disrupted gene. If the gene in question is essential. or “knock out. The gene is inactivated first and then this copy is used to generate knockout. By breeding them together.Knockout mice for medical research Transgenic animals. After the transgenic offspring are born. Once the construct is made.Knockout Mice The target gene is cloned and disrupted by inserting a DNA cassette. . This work is usually done in bacteria. two heterozygotes are crossed to create a homozygous knockout mouse. it is put back into a mouse by injection into the male pronucleus during fertilization. Location effects on expression of the transgene Transgenic organisms carrying the same inserted transgene often differ considerably in expression. Both the level of expression and the pattern of expression in various tissues of the body may vary. to construct another line of . Presence of any nearby regulatory elements like enhancers. The physical state of the DNA is important. Many of these effects are due to the location of the transgene. Insert the DNA transgenic animals. Determining the position effect Extract transgene DNA from a transgenic animal in which the transgene is not expressed. Insulator sequences or boundary elements These sequences block the activity of other regulatory elements. Site-directed insertion 1.Combating location effects on transgene expression A. Transgenes flanked by insulators probably form independent loops of DNA from which heterochromatin is excluded. . 2. Building appropriate regulatory elements in the transgene construct itself. B. Dominant control elements Some regulatory sequences control nearby genes or clusters of genes in a dominant manner. If a gene is flanked by two insulator sequences it is protected from the effects of any regulatory elements beyond the insulators. Protection of a Gene by Insulator Sequenc . The transgene is not necessarily a novel gene. and under the same regulation. Replacement of DNA . Insertional Vectors 2. as opposed to the random integration that usually happens with injected DNA. • Inserting a transgene in a specific location may be desirable for several reasons. Chromosomal location often affects the expression of a transgene. Gene replacement in the same location. 1.Targeting the transgene to a specific location • Requires homologous recombination. 2. • The DNA to be inserted is flanked by sequences that are homologous to those at the target location. 1. Targeting Vectors Rely on Homologous Recombination . cadm The heat shock promoter from Drosophila Hsp70 gene. .Deliberate control of transgene expression Inducible Endogenous Promoters Promoter is induced by heavy metals such as lead. Human-alpha-1-antitrypsin. including sheep. including insulin and many immunizations are developed in transgenic animals. For example. Transgenic pigs with human-histo-compatibility have been studied in the hopes that the organs will be suitable for transplant with less chances of rejection. pigs. Transgenic livestock have been used as bioreactors since the 1990s. which has been developed in sheep and is used in treating humans with this deficiency.Successful examples of transgenic animals. In March 2011. Stable expressions of human proteins have been developed in many animals. . the bioactive recombinant Human Lysozyme was expressed in the milk of cloned transgenic cattle. and rats. Many medicines. In 2011. Canada created the genetically engineered Enviropig.In 1999. the researchers claim these transgenic cows to be identical to regular cows. In 2009. scientists at the University of Guelph in Ontario. Aside from milk production. scientists in Japan announced that they had successfully transferred a gene into a primate species (marmosets) and produced a stable line of breeding transgenic primates for the first time. Their first research target for these marmosets was Parkinson's disease. scientists in China released news that they have introduced human genes into 300 dairy cows to produce milk with the same properties as human breast milk. This field is growing rapidly and new pharming uses are .