Transgenic Animals
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IQP-43-DSA-1172 IQP-43-DSA-2345 TRANSGENIC ANIMALS An Interactive Qualifying Project Report Submitted to the Faculty of W ORCESTER POLYTECHNIC INSTITUTE In partial fulfillment of the requirements for the Degree of Bachelor of Science By: ____________________ ____________________ Robert Brooks Nathan Levesque October 22, 2007 APPROVED: _________________________ Prof. David S. Adams, Ph.D. W PI Project Advisor 1 ABSTRACT Transgenic animals are genetically altered to express traits or behaviors not normally present in that species by inserting a foreign gene into their genome. The purpose of this IQP was to study the potential of this technology and its effects on society. The scope of our research incorporates a description of transgenic animal creation, a distribution of animal types, and a discussion of current ethical and legal debates. The conclusion formed from this research indicates that regulation and cautious deliberation of animal treatment can offer minimal animal suffering, while providing maximum benefit for society and humanity. 2 TABLE OF CONTENTS Signature Page … … … … … … … … … … … … … … … … … … … … … … … … … … … ...1 Abstract … … … … … … … … … … … … … … … … … … … … … … … … … … … ; ; ; 882 Table of Contents … … … … … … … … … … … … … … … … … … … … … … … … … … ...3 Project Objective … … … … … … … … … … … … … … ...… … … … … … … … … … … … .4 Chapter-1: Description and Construction of Transgenic Animals… … … … … … … … 5 Chapter-2: Transgenic Applications ..… … … … … … … … … … … … … … … … … … . 13 Chapter-3: Transgenic Ethics … … … … … … … … … … … … … … ..… … … … … … … 26 Chapter-4: Transgenic Legalities … … … … … … … … … … … … … … … … … … … … . 34 Conclusions … … … … … … … … … … … … … … … … … … … … … … … … … … … … ... 37 Bibliography … … … … … … … … … … … … … … … … … … .… … … … … … … … … … . 39 3 PROJECT OBJECTIVES The objective of this IQP was to examine the complexities of controversial new transgenic animal technologies, the intricacies involved with the ethics, and document the benefits or effects on society. The objective was accomplished by defining the methods for creating transgenic animals, categorizing and describing the different classes of applied technologies, and investigating the benefits to society. An analysis of the ethical and legal debates surrounding transgenic animals provided the backing for our conclusions about this technology and its role in society. 4 Chapter 1: Description and Construction Of Transgenic Animals A transgenic animal is an animal whose genome has been modified deliberately to contain a foreign gene from a different species. The genome is the genetic makeup of the animal and is responsible for its inherited characteristics. In every living organism the genome consists of DNA which is divided into genes (that code for proteins and regulatory RNAs), and some junk DNA (that seems to encode nothing useful). Genes contain information that regulates how the organism functions. Genes can be altered artificially so that some of the characteristics of an animal are changed. The genome is altered by having a gene from another source introduced into it, usually for the purpose of giving the animal a new feature, such as the ability to produce a life saving drug, or the ability to produce organs histocompatible with humans for organ transplants. Animals that have their DNA manipulated in this way are considered transgenic animals, and the purpose of this chapter is to describe how the DNA is manipulated, and how such animals are created. Creating the Transgene The first step in creating a transgenic animal is to create the DNA to be inserted. The foreign gene or genes which are inserted into an animal are called transgenes. These transgenes must be initially cloned (to make copies) then inserted into the nucleus of the egg to be transmitted through the germ line so that every cell of the animal contains the same modified genetic material. Transgenes are linear pieces of DNA made up of three 5 separate parts. The first part of the transgene is the promoter sequence. The promoter is responsible for the function of the transgene by determining the tissue in which the transgene will be expressed. The best promoter for a given transgene will depend on the exact aims of the research. For example, when creating an animal that produces a life saving drug in the milk would use the casein promoter to ensure the drug is produced only in the milk. Tissue-specific promoters can be used to limit the spatial expression pattern, while inducible promoters are used to control the timing of the expression (UCI- Transgenic Mouse Facility, 2003). The second important part of the transgene is the structural gene of interest. This part contains DNA sequences encoding the foreign protein to be expressed, such as human growth hormone or insulin. The third part of the transgene is the polyadenylation signal, also known as the termination sequence, because it dictates the end of the RNA encoding the protein. Microinjection into the Male Pronucleus Once the transgene has been cloned, it must be inserted into the genome of an animal. There are a few different ways to do this. The most popular method is to microinject the DNA into the male pronucleus. This method involves using a very fine needle (usually a thin glass hollow tube) to inject the cloned transgene into the pronucleus of a reproductive cell. The first step is to harvest freshly fertilized eggs by super-ovulating female animals by injecting them with specific hormones. These eggs are then fertilized in vitro, meaning the sperm and egg are mixed in a test tube not in the live animal. The sperm head containing the male pronucleus penetrates the egg. Before the male pronucleus fuses with the female pronucleus of the egg, the DNA is injected into 6 the male pronucleus (it is larger and easier to inject into). The injection process is performed by holding the egg with a microtube suction device and injecting a solution containing the transgene into the male pronucleus using a micropipette (Figure-1). Figure 1: DNA Microinjection into the Male Pronucleus. On the left, the microtube suction device holds the egg and the micropipette (shown on the right) inserts the transgene into the egg. (http://www.research.uci.edu/tmf/dnaMicro.htm) The fertilized and injected egg is then cultured. W hen the two pronuclei have fused to form the diploid zygote nucleus, the zygote will divide by mitosis to form a two- cell embryo, etc. The embryo is usually cultured to the blastocyst stage, about 5 days, where the embryo is a hollow ball of cells about the size of the period at the end of this sentence. The blastocyst is then implanted into the uterus of a pseudopregnant mother (prepared by mating a female mouse with a vasectomized male). W ith microinjection, the insertion of DNA is random, so no control exists as to where it inserts. If it inserts within a required gene, the process can be fatal to the animal. Following the birth of the potentially transgenic pups, they are screened to determine whether they are transgenic (discussed below). If they are transgenic, positives are often mated with other positives to increase expression of the transgene. However, the overall success rate of producing a 7 transgenic animal is very low, when using mice no more than 10-20% will have the gene, and when using farm animals no more than 0.1 œ 1.0% will be positive (Transgenic Animals, 2003). Embryonic Stem Cell Gene Transfer Another frequently used method for creating transgenic animals involves embryonic stem cell gene transfer. Embryonic stem (ES) cell lines are established from the inner cell mass of blastocysts. These ES cells can be cultured and manipulated in vitro, and will resume normal development when implanted into a recipient blastocyst. Introducing the foreign gene into these cells can be done by microinjection (as discussed above), by a virus, or using chemicals. Thus the use of these cells allows the use of a greater variety of techniques for inserting the transgene than for pronuclear micro- injection. Delivering the transgene virally is based on inserting the transgene into a virus then using the virus to infect the ES cells. Usually the virus used is mutated so it does not cause disease or multiply in infected cells. The most widely used type of virus is called a lentivirus. Lentiviruses are known as slow viruses because symptoms do no appear until long after the initial infection. These viruses (including HIV) have the ability to naturally infect both dividing and non-dividing cells, which makes them efficient for delivering genes into embryonic stem cells, and others. In developing lentiviral vectors, the DNA encoding some or all of the viral genes is removed and replaced with the foreign gene. Thus, the viral vector is designed to be able to enter the cell, deliver the gene, but does not have the ability to replicate or cause disease once inside. 8 Chemical methods have also been developed for non-viral delivery of the transgene. These methods are based on coating the DNA with lipids, polymers, or proteins. For example, micelles are formed when DNA is coated with phospholipids. The micelles can efficiently fuse with the ES cells to deliver the DNA. The lipids are useful because they are positively charged and help when the negatively charged plasmid DNA makes contact with the negatively charged cell surface. Once the specific transgene has been inserted, the ES cells containing the transgene can be selected for, which is a second strong reason for using ES cells