Because developmental biology and the use of the mouse in genetic studies have been my passion for the past two decades, my interest was immediately sparked when I laid eyes upon this book. The many years of using good old Mickey as a research tool has led to the widespread application of ever more sophisticated methods of creating genetic changes in the mouse, to, for example, follow cell behaviors, mark lineages and manipulate early developmental processes. Our final goal has always been to find phenotypes that can shed light on the intricate processes of normal development and– if something goes wrong – on disease processes. It was not surprising to see that both of the authors of this book are teaching at the Cold Spring Harbor course `Molecular Embryology of the Mouse'. These yearly events, where mouse embryology and genome manipulation techniques are taught, are perhaps the most comprehensive learning opportunities for anyone interested in entering this exciting field. The next step in the process, however – following the tedious road of analyzing the phenotypes we have created – has thus far not been matched by any course or reference book. This new publication, Mouse Phenotypes: A Handbook of Mutation Analysis, is meant to fill this gap. The authors have chosen the motto of their book very well, quoting our much-admired `mother' of mouse developmental genetics, Anne McLaren: `A mouse without a phenotype is non-existent'.
My journey through the chapters began with how to design a construct to generate mutations, and continued all the way to the completed analysis of complex and subtle phenotypes. However, this book is not meant to be read straight through from page 1 to page 235. Neither is it a collection of independent compartments. Instead, the chapters are linked with a roadmap, and I am directed to the exact path relevant to my particular situation simply by answering a few key questions at each critical step. Clear signposts guide me through the jungle of methods and swamps of possible pitfalls, making sure that I spend my time and resources on the most relevant experiments. To make this book complete, it is sprinkled full with helpful hints and tips on how to avoid costly mistakes. If you were expecting to find protocols for how to perform various assays, you will be disappointed. However, you will soon realize that adding protocols would have been a mistake: they could not possibly be fitted into one book. Instead, the authors have taken a wise approach by providing references to literature on versatile hands-on protocols. Having said this, you may expect the book to be heavy on theoretical reading – wrong again! The layout provides an excellent overview, and the language is very pleasant and easy to read. The text is to the point, without too much history and with just enough background to make it comprehensive.
For a newcomer to the field of mouse genetics, this book is a must. Those who have once burned their fingers in the process of mouse mutation analysis will get the feeling of `oh yeah, now I know what I did wrong last time' and will be encouraged to go ahead and try again. For those of us who `have been there, seen that', it offers a wealth of new ideas for alternate approaches. The third edition of Manipulating the Mouse Embryo (Nagy et al., 2003) is a logical companion to this book, and it is not surprising to see that many references are made to protocols that can be found there. The intention to keep the text `clean' by avoiding references is good, but occasionally I got lost when searching for specific data. For example, when looking for chromosomal segments in the genome of the 129 mouse strain that might be difficult to target, or for the mutant variant of tk that does not cause male sterility, I am guided to Appendix 1. Once in the appendix, I find myself lost as to where to retrieve the information I am looking for.
The first two chapters contain the all-important theory. The focus is on practical advice for making every project a success. First, it describes the various types of methods for generating mutants: chemical and X-ray induced mutagensis, gene-trap approaches, retroviral infections, transposons and classical transgenics. The second chapter gives a detailed analysis of the practical sides of gene targeting strategies, and lists the pros and cons of different methods. A step-by-step guide took me through the process, leaving out no important considerations. Positive and negative selection markers, how best to make a null allele, point mutations, conditional alleles and reporter `knock-ins' are all discussed.
In the following two chapters, the time has come to actually make a mouse that carries a specific mutation. Because the most versatile (and therefore also often the most challenging) approach requires the use of embryonic stem (ES) cells, the generation of chimeras and germline transmission, these chapters heavily focus on these techniques. Here, I got answers to all those questions that often remain after I've read the literature, tried the assays and found that nothing seems to work. The authors describe the possible reasons as to why a genetargeting experiment might not be working and suggest trouble-shooting techniques for tackling problems such as no correctly targeted clones, no chimeras, no offspring from chimeras or no germline transmission. Once a mutation has successfully been passed through the germline, the task of maintaining the mouse line begins, and this book offers detailed discussions of important topics, such as genetic background modifier effects, backcrossing schemes to obtain coisogenic lines, how to delete selection cassettes in vivo and special techniques for dealing with infertility phenotypes. The authors provide many useful suggestions for how to test for genetic interactions and for redundancy between members of a gene family. I found only some minor omissions, such as the possible `tricolor' phenotype of E14TG2 ES cell line chimeras (depending on the host embryo used to make them, the ES-derived fur is not always pale yellow), and I would have liked a more extensive discussion of embryo transfer as a means for rederiving pathogen-loaded lines.
Chapters 5 and 6 are the ones most readers will probably be looking for, as they discuss the analysis of phenotypes that manifest at pre- and postnatal stages. Also here are lots of practical tips for avoiding common mistakes, and for performing tricky analyses with very limited resources by using innovative simple techniques. One such example is how to reliably count and image the blastomeres in late pre-implantation-stage embryos. The text is complete, covering important steps such as failure of the embryo to divide, hatch or implant (including in vitro assays for assessing the developmental potential of trophoblast and inner cell mass cells). The book then turns to looking at postnatal phenotypes. This section starts with a discussion of perinatal lethality and its possible causes, such as developmental delay, cranial nerve, cardiovascular, skeletal and diaphragm defects, and cleft palate. For each case, the authors offer advice on which organ system is likely to be affected, and on how to go about further and deeper analysis. With the same systematic precision, they take the reader on the quest of finding postnatal/pre-weaning phenotypes – both lethal and subtler ones. Finally, we arrive at the much dreaded `no phenotype' finding. The authors point out quite correctly that there is no such thing – we just have not looked carefully enough! So, they give further advice on how to do a proper search by using, for example, neurological and behavioral tests, genetic background effects and environmental challenges.
Chapter 7 deals with dominant effects and offers advice on how to deal with the special challenges these situations may present in ES cells, chimeras and heterozygous offspring. Dominant reproductive problems are not forgotten, and the mechanisms behind both dominant and semi-dominant effects are discussed in detail, including haploinsufficiency, and mutations in X-linked, Y-linked and imprinted genes.
The last chapter re-visits both Chapters 2 and 5 in that it deals with early lethal phenotypes. The usefulness of conditional and inducible alleles now becomes fully clear, and the book offers advice on how to test for and use these methods. The authors then describe sophisticated methods of using different kinds of chimeras as a means of rescuing and analyzing otherwise `impossible to reach' phenotypes, such as the use of tetraploid embryos in combination with ES cells. However, I was surprised that a discussion of the advantages of using F1 hybrid cell lines for this purpose is missing from this chapter.
Two very well-thought through appendices bring the book to an end. In the first, I can find useful literature and web-based resources; the second provides a list of published phenotype analyses that give good examples of the work that lies ahead for the reader.
It is not difficult to imagine why no author has thus far attempted to take the very ambitious challenge of writing a book such as this. Richard Behringer and Virginia Papaioannou have accomplished this task in a truly impressive manner. If you are a student or researcher entering the field of mouse genetics, the money you invest in acquiring this book will be worth every penny. The book will provide you with a guided tour of how to make the most complete analysis of every possible phenotype you may stumble upon. While holding onto the authors' hands through this maze, you can rest assured that you will have exploited all available possibilities, while at the same time not having wasted energy on working towards dead ends. To put it simply, this book `puts it all together'. It is truly a work we all have been waiting for!
- © 2005.