Introduction to Transgenic Animal Technology

Carl A. Pinkert,    Department of Biological Sciences, College of Arts and Sciences, The University of Alabama, Tuscaloosa, AL

Transgenic animals continue to embody one of the most potent and exciting research tools in the biological sciences. Transgenic animals represent unique models that are custom tailored to address specific biological questions. Hence, the ability to introduce functional genes into animals provides a very powerful tool for dissecting complex biological processes and systems. Gene transfer is of particular value in those animal species, where long life cycles reduce the value of classical breeding practices for rapid genetic modification. The third edition of Transgenic Animal Technology updates the prior two editions with current techniques, instrumentation, and technologies.

Keywords

Transgenic animals; animal transgenesis; genetic engineering; animal biotechnology

I Introduction

The last three decades witnessed a rapid advance of the application of genetic engineering techniques for increasingly complex organisms, from single-cell microbial and eukaryotic culture systems to multicellular whole-animal systems. The whole animal is generally recognized as an essential tool for biomedical and biological research, as well as for pharmaceutical development and toxicological/safety screening technologies. Moreover, an understanding of the developmental and tissue-specific regulation of gene expression is achieved only through in vivo whole-animal studies.

Today, particularly with gene editing technologies on the rise, transgenic animals (and animal biotechnologies) continue to embody one of the most potent and exciting research tools in the biological sciences. Transgenic animals represent unique models that are custom tailored to address specific biological questions. Hence, the ability to introduce functional genes into animals provides a very powerful tool for dissecting complex biological processes and systems. Gene transfer is of particular value in those animal species where long life cycles reduce the value of classical breeding practices for rapid genetic modification. For identification of interesting new models, genetic screening and characterization of chance mutations remain a long and arduous task. Furthermore, classical genetic monitoring cannot adequately engineer a specific genetic trait in a directed fashion.

II Historical Background

In the early 1980s, only a handful of laboratories possessed the technology necessary to produce transgenic animals. With this in mind, this text is envisioned as a bridge to the development of various transgenic animal models. Hopefully, through the first two editions the curiosity and interests of researchers in diverse research fields were influenced productively. The gene transfer technology that is currently utilized across vertebrate species was pioneered using mouse and domestic animal models. Today, the mouse continues to serve as a starting point for implementing a variety of gene transfer procedures and is the standard for optimizing experimental efficiencies for many species. Inherent species differences are frequently discounted by researchers who are planning studies with a more applicable species model. However, when one attempts to compare experimental results generated in mice to those obtained in other species, not surprisingly many differences become readily apparent. Therefore, an objective of this text will be to address the adaptation of relevant protocols.

When initiating work related to gene transfer, it is important to look at the rapid advancement of a technology that is still primitive by many standards. From an historical perspective, one readily contemplates potential technologies and methods that lie just ahead. Whereas modern recombinant DNA techniques are of primary importance, the techniques of early mammalian embryologists were crucial to the development of gene transfer technology. While we can look at well over three decades of transgenic animal production, the preliminary experiments leading to this text go back millennia to the first efforts to artificially regulate or synchronize embryo development. Amazingly, it has been more than a century since the first successful embryo transfer experiments, dating back to the efforts first published in the 1880s and to Heape’s success in 1891 (Heape, 1891). By the time the studies by Hammond were reported in the late 1940s, culture systems were developed that sustained ova through several cleavage divisions. Such methods provided a means to systematically investigate and develop procedures for a variety of egg manipulations. These early studies led to experiments that ranged from mixing of mouse embryos and production of chimeric animals, to the transfer of inner cell mass cells and teratocarcinoma cells, to nuclear transfer and the first injections of nucleic acids into developing ova. Without the ability to culture or maintain ova in vitro, such manipulations or the requisite insights would not be possible (Brinster and Palmiter, 1986).

Gurdon (1977) transferred mRNA and DNA into Xenopus eggs and observed that the transferred nucleic acids could function in an appropriate manner. This was followed by a report by Brinster et al. (1980) of similar studies in a mammalian system, using fertilized mouse ova in initial experiments. Here, using rabbit globin mRNA, an appropriate translational product was obtained.

Major turning points in science continue to accelerate at an incredible pace. The technology available in 1994 had developed considerably and, as predicted, a number of areas from both the first and second editions of this text are clearly antiquated or obsolete today. It is amazing to look back at the major events related to genetic engineering of animals and how our ability to manipulate both the nuclear and mitochondrial genomes has come so far.

The production of transgenic mice has been hailed as a seminal event in the development of animal biotechnology. In reviewing the early events leading to the first genetically engineered mice, it is fascinating to note that the entire procedure for DNA microinjection was described nearly 50 years ago. While some progress seems extremely rapid, it is still difficult to believe that, following the first published report of a microinjection method in 1966 (Lin, 1966; Figures 1.1 and 1.2), it would be another 15 years before transgenic animals were created. The pioneering laboratories that reported success at gene transfer would not have been able to do so were it not for the recombinant DNA technologies necessary to develop protocols or document results (Gordon et al., 1980; Wagner et al., 1981a,b; Harbers et al., 1981; Brinster et al., 1981; Costantini and Lacy, 1981; Gordon and Ruddle, 1981). In gene transfer, animals carrying new genes (integrating foreign DNA segments into their genome) are referred to as transgenic, a term first coined by Gordon and Ruddle (1981). As such, transgenic animals were recognized as specific variants of species following the introduction and/or integration of a new gene or genes into the genome. As for many technologies, the definition of transgenic animals has taken on a broader meaning and perspective that is more inclusive and includes animals either integrating foreign DNA segments into their genome following gene transfer or resulting from the molecular manipulation of endogenous genomic DNA (Pinkert et al., 1995). Yet, as outlined by Beardmore (1997), this definition, too, required refinement as “state-of-the-art” technologies continue to evolve. And yes, we now include both the nuclear genome as well as the mitochondrial genome with the inclusion of transmitochondrial animal modeling first described in the mid-1990s (where we coined the term mitomice in 1997, see Wawrousek, 1998) (Dunn et al., 2012; Irwin et al., 2013).

Through the years, there have been literally thousands of excellent reviews that detail the production of transgenic animals, in addition to a journal, Transgenic Research, dedicated to this field. In the first edition, readers were referred to now classical reviews by Brinster and Palmiter (1986), Bürki (1986), Camper (1987), Cordaro (1989), First and Haseltine (1991), Grosveld and Kollias (1992), Hogan et al. (1986), Palmiter and Brinster (1986), Pattengale et al. (1989), Pinkert (1987), Pinkert et al. (1990), Pursel et al. (1989), Rusconi (1991), Scangos and Bieberich (1987), and Van Brunt (1988). However, to this day, in my opinion, the singular effort with the greatest...