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Using CRISPR-based genome-editing techniques, we are able to generate a variety of new mouse models of several types of diseases. These animal models will be instrumental not only for enabling the comprehension of a particular disease and its underlying molecular mechanism but also as unique recipients for testing novel and innovative therapeutic approaches that are being currently explored. This chapter describes detailed step-by-step protocols, reagents, and equipment required for successful generation of genome-edited mice using CRISPR tools.

Mice provide an unlimited source of animal models to study mammalian gene function and human diseases. The powerful genetic modification toolbox existing for the mouse genome enables the creation of, literally, thousands of genetically modified mouse strains, carrying spontaneous or induced mutations, transgenes or knock-out/knock-in alleles which, in addition, can exist in hundreds of different genetic backgrounds. Such an immense diversity of individuals needs to be adequately annotated, to ensure that the most relevant information is kept associated with the name of each mouse line, and hence, the scientific community can correctly interpret and benefit from the reported animal model. Therefore, rules and guidelines for correctly naming genes, alleles and mouse strains are required. The Mouse Genome Informatics Database is the authoritative source of official names for mouse genes, alleles, and strains. Nomenclature follows the rules and guidelines established by the International Committee on Standardized Genetic Nomenclature for Mice. Herewith, both from the International Society for Transgenic Technologies (ISTT) and from the scientific journal Transgenic Research, we would like to encourage all our colleagues to adhere and follow adequately the standard nomenclature rules when describing mouse models. The entire scientific community using genetically modified mice in experiments will benefit.

The generation of transgenic mice by microinjection of DNA into the pronuclei of fertilized oocytes was described in the early 1980s. A number of parameters affecting the efficiency of the technique were soon identified, including the type of DNA construct, the concentration of DNA being injected, and, most importantly, the strain of mice used for oocyte donors. Since then, hundreds of laboratories and transgenic core facilities across the world have successfully used this technique, essentially as originally described, to create thousands of new transgenic mouse lines. However, the overall procedure continues to be relatively inefficient, in terms of the number of fertilized oocytes required to produce a transgenic mouse, and variations in yields from day to day and construct to construct can be large. Consequently, core facilities often struggle to explain to their customers why a sufficient number of transgenic founders were not produced from a given construct. We believe the field (and individual facilities) would benefit from a rigorous assessment of average yields and expected variations in yields. To this end, we have initiated a survey from the International Society for Transgenic Technologies (ISTT) web site ( www.transtechsociety.org ), to obtain raw microinjection data from as many facilities as possible. We intend to use this data to establish performance standards for the field. Existing facilities will be able to refer to these standards in dealing with dissatisfied clients, and new facilities will be able to aim for an achievable goal. We may even be able to discover an optimum combination of factors that will allow every facility to achieve higher yields.

The production of animals with large transgenes is an increasingly valuable tool in biotechnology and for genetic studies, including the characterization and manipulation of large genes and polygenic traits. In the present study, we describe an intracytoplasmic sperm injection (ICSI) method for the stable incorporation and phenotypic expression of large yeast artificial chromosomes (YAC) constructs of submegabase and megabase magnitude. By coinjecting spermatozoa and YACs into metaphase II oocytes, we were able to produce founders exhibiting germline transmission of an intact and functional transgene of 250 kilobases, carrying the mouse tyrosinase locus, used here as a reporter gene to rescue the albinism of recipient mice. More than 35% transgenesis was obtained for this YAC transgene. When compared with the pronuclear microinjection standard method, the efficiency of the ICSI-mediated YAC transfer system was significantly greater. In summary, we describe, for the first time, stable incorporation in the host genome and correct phenotypic expression of large DNA constructs mediated by ICSI.

The generation and analysis of transgenic mice has become an important tool to progress our understanding of human and mouse gene function and its association with human genetic diseases. Animal models, based on genetically modified mice, both standard transgenic and knock-out animals, are increasingly being used worldwide. Monitoring of transgenic mouse production and transgenic mouse colonies is required to efficiently manage the resources that are available. Here, I describe three independent FileMaker databases (transgenics, mymouse and cages) that have been developed to track the generation of transgenic mice, the organisation of transgenic mouse colonies and the distribution of mice in cages. These three databases are freely available for academic use.

Pigmentary genes were among the first mammalian genes to be studied, mostly because of the obvious phenotypes associated with their mutations. In 1990, tyrosinase, encoding the limiting enzyme in the melanin synthesis pathway, was eventually assigned to the c (albino) locus by classical rescue experiments driven by functional constructs in transgenic mice. These pioneer reports triggered the study of the regulation of endogenous tyrosinase gene expression by combining different amounts of upstream regulatory and promoter regions and testing their function in vivo in transgenic animals. However, faithful and reproducible transgenic expression was not achieved until the entire tyrosinase expression domain was transferred to the germ-line of mice using artificial-chromosome-type transgenes. The use of these large tyrosinase transgenic constructs and the ease with which they could be manipulated in vitro enabled the discovery of previously unknown but fundamental regulatory regions, such as the tyrosinase locus control region (LCR), whose presence was required in order to guarantee position-independent and copy-number-dependent expression of tyrosinase transgenes, with an expression level, per copy, comparable to that of an endogenous wild-type allele. Subsequently, functional dissection of elements present within this LCR through the generation of new artificial-chromosome type tyrosinase transgenes has revealed the existence of different regulatory activities. The existence of some of these units had been suggested previously by standard-type transgenic analyses. In this review, we will discuss both independent approaches and conclude that optimal tyrosinase transgene expression requires the use of its complete expression domain.

FVB/N mice are one of the most common inbred strains for the generation of transgenic animals. This mouse strain is preferred for transgenesis because of its fertilized oocytes, which have unique pronuclei for microinjection, and its vigorous reproductive performance along with consistently large litter sizes. However, these inbred mice carry a retinal degeneration mutation caused by a proviral insertion into the Pdeb gene, encoding the beta subunit of cGMP phosphodiesterase. This mutation (Pdeb(rd1), formerly known as rd) results in postnatal rod photoreceptor degeneration and causes severe visual impairment, which may be relevant for behavioural and vision-related research. This deficit can be overcome by crossing these mice with other mouse strains carrying the wild-type allele at the Pdeb locus. We have devised a simple polymerase chain reaction (PCR)-based method for distinguishing between the mutant and the wild-type alleles, thus allowing the efficient monitoring of the Pdeb(rd1) mutation in FVB/N-derived transgenic mice prior to experimentation where visual deficit is expected to have an influence in the phenotype.

Several groups have recently reported the successful generation of transgenic mice harbouring yeast artificial chromosomes (YACs). Different methodological approaches have been shown to produce similar results, namely, the faithful expression of the transgenes carried on YAC DNA. In this paper, we compare the reported techniques for obtaining transgenic mice carrying YACs using a 250-kb YAC bearing the mouse tyrosinase gene. These methods include: microinjection of gel-purified YAC DNA into pronuclei of fertilized mouse oocytes, yeast spheroblast fusion with embryonic stem (ES) cells and lipofection of YAC DNA into ES cells. Taken together, these reports show that the delivery of large genomic regions covering a gene of interest (such as those cloned in YAC vectors) is feasible, and will ensure appropriate temporal and spatial expression of the transgene at a level comparable to that of the endogenous counterpart.

Yeast artificial chromosomes (YACs) represent the latest generation of vectors which have the great advantage of large insert size. The introduction of YACs into mammalian cells and organisms has become an important goal, since it offers the potential to study the control of large and complex transcription units and identify genes by complementation. Microinjection into the nucleus is the most direct and efficient way of delivering YAC DNA into cells, but requires the purification of the YAC from the remaining yeast chromosomes. Here we describe a detailed method for the isolation of pure, intact and highly concentrated YAC DNA. As a model system the murine tyrosinase gene was chosen and four YACs covering this locus were isolated. Introduction by homologous recombination in yeast of sequences permitting YAC amplification greatly facilitated the isolation of YAC DNA at high concentrations. YAC DNA stabilized in a salt and polyamine containing buffer did not compromise the survival of microinjected oocytes and was suitable for the generation of transgenic mice. Applications and benefits of this technique will be discussed.

Transgenic mice have become invaluable for analysing gene function and regulation in vivo. However, the size of constructs injected has been limited by the cloning capacity of conventional vectors, a constraint that could be overcome with yeast artificial chromosomes (YACs). We investigated the feasibility of making transgenic mice with YACs by pronuclear injection of a small YAC carrying a gene encoding tyrosinase. Use of a vector with a conditional centromere allowed fifteenfold amplification of the YAC in yeast and its recovery in high yield. The albino phenotype of the recipient mice was rescued demonstrating the correct expression of the tyrosine gene from the construct. Furthermore, the telomeric sequences added by the yeast integrated into the mouse genome and did not reduce efficiency of integration. Using this technique future experiments with longer YACs will allow the expression of gene complexes such as Hox and the globin gene clusters to be analysed in transgenic animals.