Melina R. Kibbe and Scott A. LeMaire (eds.)Success in Academic SurgerySuccess in Academic Surgery: Basic Science201410.1007/978-1-4471-4736-7_12© Springer-Verlag London 2014
12. Use of Genetically Engineered Mice for Research
(1)
Department of Surgery, University of Pittsburgh Medical Center, F677 Presbyterian Hospital, 200 Lothrop Street, Pittsburgh, PA 15213, USA
(2)
Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, University of Pittsburgh Medical Center, Montefiore Hospital, 7 South, 3459 Fifth Avenue, Pittsburgh, PA 15213, USA
Abstract
Constructing a unique animal model that closely mimics a surgical disease of interest is an important step for researchers when embarking on a career in basic science research. The mouse is an excellent experimental model for defining human gene function because of the genetic similarity to humans and ease of manipulation by molecular means. Genomic sequencing has demonstrated that of ~30,000 genes in both mice and humans, only 1 % is unique to either species (Doyle et al., Transgenic Res 21(2):327–349, 2012). Despite this similarity, the creation of a significant number of genetically altered mice with the same phenotype can be a difficult task. Nevertheless, the use of genetically engineered mice has been, and continues to be, a powerful tool for studying human disease and developing therapeutics. The use of transgenic and knockout mice for research allows investigators to study gene function, dissect molecular mechanisms, and define cellular pathways underlying disease states. In order to accomplish this, consideration must be given to the pathophysiology of the disease to be modeled. A specific understanding of how the disease develops will affect which animal model will be best suited for one’s research investigations. By manipulating the mouse genome, researchers are able to accelerate the research process, discover novel targets, and make further contributions to various fields of research. The objective of this chapter is to provide an overview of the creation of common mouse models of human disease and to serve as a resource for identifying specific mouse strains that may be used for research investigations.
Keywords
TransgenicKnockoutKnock inCre recombinaseGene targetingGenetic engineeringIntroduction
Constructing a unique animal model that closely mimics a surgical disease of interest is an important step for researchers when embarking on a career in basic science research. The mouse is an excellent experimental model for defining human gene function because of the genetic similarity to humans and ease of manipulation by molecular means. Genomic sequencing has demonstrated that of ~30,000 genes in both mice and humans, only 1 % is unique to either species [1]. Despite this similarity, the creation of a significant number of genetically altered mice with the same phenotype can be a difficult task. Nevertheless, the use of genetically engineered mice has been, and continues to be, a powerful tool for studying human disease and developing therapeutics. The use of transgenic and knockout mice for research allows investigators to study gene function, dissect molecular mechanisms, and define cellular pathways underlying disease states.
In order to accomplish this, consideration must be given to the pathophysiology of the disease to be modeled. A specific understanding of how the disease develops will affect which animal model will be best suited for one’s research investigations. By manipulating the mouse genome, researchers are able to accelerate the research process, discover novel targets, and make further contributions to various fields of research. The objective of this chapter is to provide an overview of the creation of common mouse models of human disease and to serve as a resource for identifying specific mouse strains that may be used for research investigations.
Transgenic Mice
By definition, transgenesis is the introduction of DNA from one species into the genome of another species [2]. Transgenic mice contain foreign genetic material integrated into the genome of every cell [3]. The foreign gene is constructed using recombinant DNA technology. This piece of DNA includes the structural gene of interest, a promoter and enhancer to allow the gene to be expressed, and vector DNA to enable transgene insertion into the mouse genome [2]. In addition to what is produced endogenously, successful integration of this DNA results in the overexpression of the transgene, with the net effect being a gain of function of the protein of interest.
How Are Transgenic Mice Produced?
Transgenic mice are produced through a process of embryonic microinjection. Foreign DNA is injected into the pronucleus of a fertilized embryo and then transferred into a recipient female mouse, allowing for random integration into the host genome [4]. Potential transgenic mice are born and identified by the polymerase chain reaction (PCR) assay. The heterozygous offspring are then bred to wild-type mice to produce heterozygous siblings that are mated to each other to create homozygous transgenic strains, which can then be used for research experiments. The number of integrated transgenes is generally inversely proportional to fragment size [3]. Thus, the copy number will be smaller with a larger transgene. When using this technique, it is important to have a thorough understanding of the target gene structure to ensure adequate protein expression in order to yield the best results. Transgenic mice can also be produced by embryonic stem cell-mediated gene transfer; a similar technique is used for the production of knockout mice and will be described in more detail below.
Advantages/Disadvantages of Using Transgenic Mice for Research
Transgenic experiments can be used by researchers in a variety of ways. The development of the transgenic mouse allows researchers to mimic human disease in a laboratory animal. The hope is that by better understanding how a certain gene contributes to a particular disease, researchers can then discover drugs that either act directly on that gene or alter its mechanism of action. Once novel targets and therapeutic strategies are developed, they can be tested in vivo before moving onto human trials.
Transgenic animals also provide the means for researchers to learn about a disease state, understand the capacity of some genes to induce specific developmental changes, demonstrate the oncogenic nature when they are expressed aberrantly, and help to find a cure that corrects the mutation responsible for the disease. Transgenic animals provide the additional benefit of affording a safe and controlled manner to test potential treatments. When embarking on a research career, transgenic mice can be extremely useful in the experimental design since transgenics can be applied to a wide range of pathologies ranging from age-related diseases to cancer.
Despite the benefits of such transgene technology, there are drawbacks. This is a time consuming, expensive, and technically demanding process. It is generally accepted that a minimum of 15–17 weeks is required from DNA injection until a transgene from a founder mouse is established in multiple mice [5]. If the transgene is injected randomly into the mouse genome by embryo microinjection, the DNA can integrate into nonspecific loci during cell division and result in deadly mutations and/or toxicity related to multiple insertions, complex rearrangements, and excessive overexpression. In approximately 5–10 % of all cases, homozygosity for a particular transgene locus has been found to cause lethality or some other phenotypic anomaly [6].
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