Transgenic Animal Technology

A Laboratory Handbook

Carl A. Pinkert

• Publisher: Elsevier
• Year: 2014
• ISBN:
  9780124095366
• Edition: 3
• Topic: Biological Sciences
• Subtopic: Zoology
• Index: Biological Sciences

Transgenic animal technologies and the ability to introduce functional genes into animals have revolutionized our ability to address complex biomedical and biological questions. This well-illustrated handbook covers the technical aspects of gene transfer — from molecular methods to whole animal considerations — for important laboratory and domestic animal species. It describes methodologies as employed by leading laboratories and is a key resource for researchers, as well as a tool for training technicians and students. This second edition incorporates updates on a variety of genetic engineering technologies ranging from microinjection and ES cell transfer to nuclear transfer in a broad range of animal modeling systems.

• Contains a comprehensive collection of transgenic animal and gene transfer methods
• Discusses background and introduction to techniques and animal systems
• Teaches practical step-by-step protocols
• Fully revised with updates to reflect state-of-the-art technology and associated changes to date

Section Four

Molecular Biology, Analyses and Enabling Technologies

Outline

16 Analysis of Phenotype

17 Design of Vectors for Optimizing Transgene Expression

18 Analysis of Transgene Integration

19 PCR Optimization for Detection of Transgene Integration

20 Analysis of Transgene Expression

21 Compound Transgenics: Recombinase-Mediated Gene Stacking

22 Assisted Reproductive Technologies and Embryo Culture Methods for Farm Animals

23 Modifying Mitochondrial Genetics

24 Databases, Internet Resources, and Genetic Nomenclature

16

Analysis of Phenotype

Cory F. Brayton¹, Colin McKerlie² and Steve Brown³, ¹Johns Hopkins University, Baltimore, MD, ²The Hospital for Sick Children and University of Toronto, Toronto, ON, Canada, ³MRC Mammalian Genetics Unit, Harwell, Oxford, UK

Phenotyping of genetically engineered animals is done to provide data and insight about the functions of genes. In academic hypothesis-driven research settings, phenotyping tests are limited to those that answer-specific questions. In large-scale omics (phenomics) programs like the International Mouse Phenotyping Consortium (IMPC), initial or primary phenotyping aims to be unbiased and broadly informative, systematic and standardized, subject to quality assurance (QA) and quality control (QC), and responsive to ongoing assessment and revision. Genetically engineered mice (GEM) or mouse embryonic stem (ES) cell lines, protocols, and data are publicly accessible from these international programs. These resources need to be user-friendly to optimize the utility of GEM models of human disease in translational research. In phenotyping and in other translational research involving animals, pathology is a critical aid to diagnose health problems that can compromise the research, and also is a powerful tool to identify, confirm, and characterize phenotypes, validate translational models, and provide biologically-relevant morphological data in the context of the whole animal. This chapter discusses phenotyping sensu latu, but emphasizes IMPC’s 10-year plan to phenotype every protein-coding gene, as well as to develop practical pathology to support phenotyping and other translational research.

Keywords

Genetically engineered mice (GEM); International Knockout Mouse Consortium (IKMC); International Mouse Phenotyping Consortium (IMPC); mice; pathology; phenotype; phenotyping; transgenic

I Introduction

The term phenotyping is used frequently in functional genomics research and literature to refer to assessing phenotypes (features, traits, abnormalities) in genetically engineered animals. In this context it is done to provide data and insight about the functions of genes, or gene products in a living system (Brown and Moore, 2012a,b). Tests used for phenotyping are used to diagnose disease, or to assess responses to therapeutic interventions in clinical settings, or are used to assess responses to experimental interventions in other preclinical research settings. Their use is not restricted to phenotyping of genetically engineered mice (GEM). High-throughput-testing pipelines, developed by the International Mouse Phenotyping Consortium (IMPC), aim to be efficient and broadly informative to diverse research areas. This chapter discusses phenotyping sensu lato, but highlights tests and resources included in the IMPC’s 10-year plan to phenotype every protein-coding gene, as well as developing practical pathology to support phenotyping and other translational research. Depending on the aims and research settings, phenotyping tests and strategies vary widely, but the approaches can be characterized as primarily: (1) hypothesis-driven, (2) purpose-driven, or (3) hypothesis-generating, or some combination of these.

A Hypothesis-Driven (or Hypothesis-Testing) Phenotyping

Hypothesis-driven (or hypothesis-testing) phenotyping aims to test specific hypotheses. Simple and specific, robust tests are favored by editors, reviewers, and granting agencies. This approach continues to dominate academic research, where GEM phenotyping correlates with the aims, hypotheses, and resources of the investigator. Thus phenotyping strategies, methodology, terminology, and the “nature and nurture” of the animals tested (their genetic backgrounds, diet, microbial status, and other housing and test conditions), vary substantially by laboratory. The advantage of this approach is that it should answer an important question without wasting resources. Disadvantages include difficulties in comparing or replicating studies from different laboratories (Crabbe et al., 2006; Wahlsten et al., 2006). Studies that cannot be replicated are justifiably criticized and contribute to concerns about failures of animal models to translate to human conditions or therapies (Kilkenny et al., 2009, 2010; ILAR-NRC, 2011). Also, the limited breadth of the phenotyping, while sufficient to test the hypothesis, may sacrifice opportunities to identify, characterize, and associate additional phenotype traits resulting from unrecognized gene pleiotropy.

B Purpose-Driven Phenotyping

Purpose-driven phenotyping generates data sets relevant to a specific area of investigation. In pharmaceutical settings such data sets are collected for the purpose of determining or defining the safety, toxicity, and carcinogenicity of a compound or device. Final testing is conducted according to US Food and Drug Administration (FDA) Good Laboratory Practices (GLP) or other international standards, and is subject to extensive quality assurance (QA) and quality control (QC) measures. Protocols, procedures, terminology, and reporting tend to be comprehensive and standardized. Examples of these types of protocols, study design, and results are available on line from the National Toxicology Program (NTP) (http://ntp.niehs.nih.gov/). Best practices, diagnostic criteria, and terminology are reviewed and updated periodically (Crissman et al., 2004). Influences of diverse microbial, dietary, and other factors on study results have been discussed (Haseman et al., 1989; Ward et al., 1994; Hailey et al., 1998; Rao and Crockett, 2003; Martin et al., 2010), as has the utility of historical control data (Elmore and Peddada, 2009; Keenan et al., 2009a,b). Commercial producers of research animals also may collect data sets on growth, longevity, fertility, and production (phenotypes), sometimes clinical chemistry and pathology, as well as on QA and QC procedures such as microbial surveillance or genetic monitoring, for the purpose of characterizing (and selling) their products (mice), or other research animals. The information can provide useful benchmarks and husbandry suggestions for maintenance and breeding, or baseline data to guide experimental design.

C Systematic (Hypothesis-Generating) Phenotyping

Systematic (hypothesis-generating) phenotyping is exemplified by the IMPC, from which broad based, unbiased, and standardized phenotyping is expected to provide insights across many areas of biology to expose new functions of well-characterized genes and offer insight to genes with little or no known function (Gailus-Durner et al., 2009; Abbott, 2010; Guan et al., 2010; Moore and IMPC and SteeringCommittee, 2010; Nature, 2010; Wurst and de Angelis, 2010; Brown and Moore, 2012a,b).

Beginning in 2002, the EUMORPHIA program developed standard operating procedures for comprehensive, high throughput primary phenotyping pipelines termed EMPReSS (European Mouse Phenotyping Resource of Standardized Screens), and a database for EMPReSS data called EuroPhenome (http://www.europhenome.org/). From these efforts, the EUropean MOuse DIsease Clinic (EUMODIC) consortium (http://www.eumodic.org/) was developed to conduct primary phenotyping of 500 mutant mouse lines produced from the C57BL/6N ES cell resource generated by the International Knockout Mouse Consortium (IKMC; http://knockoutmouse.org) (Brown et al., 2005; Hrabe de Angelis et al., 2006). The EUMODIC consortium of four sites with expertise in mouse genetics, functional genomics, and analysis conducted the pilot studies from which the IMPC phenotyping pipeline was developed and is now implemented at 13 phenotyping centers within the consortium (http://www.mousephenotype.org/).

IMPC pipeline and protocol information is available through IMPReSS (International Mouse Phenotyping Resource of Standardized Screens, formerly EMPReSS) https://www.mousephenotype.org/impress/. Data are generated and quality-reviewed by each center, uploaded to the IMPC Data Coordinating Cent...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• List of Contributors
• Preface
• Section One: Overview
• Section Two: Transgenic Animal Production Focusing on the Mouse Model
• Section Three: Production of Transgenic Laboratory and Domestic Animal Species
• Section Four: Molecular Biology, Analyses and Enabling Technologies
• Glossary