Marcello Raspa

EMMA—mouse mutant resources for the international scientific community

The laboratory mouse is the premier animal model for studying human disease and thousands of mutants have been identified or produced, most recently through gene-specific mutagenesis approaches. High throughput strategies by the International Knockout Mouse Consortium (IKMC) are producing mutants for all protein coding genes. Generating a knock-out line involves huge monetary and time costs so capture of both the data describing each mutant alongside archiving of the line for distribution to future researchers is critical. The European Mouse Mutant Archive (EMMA) is a leading international network infrastructure for archiving and worldwide provision of mouse mutant strains. It operates in collaboration with the other members of the Federation of International Mouse Resources (FIMRe), EMMA being the European component. Additionally EMMA is one of four repositories involved in the IKMC, and therefore the current figure of 1700 archived lines will rise markedly. The EMMA database gathers and curates extensive data on each line and presents it through a user-friendly website. A BioMart interface allows advanced searching including integrated querying with other resources e.g. Ensembl. Other resources are able to display EMMA data by accessing our Distributed Annotation System server. EMMA database access is publicly available at http://www.emmanet.org.

Helicobacter typhlonius was detected in the sex organs of three mouse strains but did not transmit vertically

Helicobacter typhlonius, one of the most recently identified members of this rapidly expanding genus of bacteria, was detected by polymerase chain reaction (PCR) in an animal facility and studied for tissue distribution in sentinel mice (Helicobacter-free, barrier maintained). Immunodeficient athymic nude-nu (nu/nu), Helicobacter-sensitive C3H/HeJ and Helicobacter-resistant C57BL/6J mouse strains were used to study whether Helicobacter species could spread to the sex organs and be transmitted vertically. Sentinel mice of these three strains became infected at different times after first exposure and Helicobacter was detected by PCR for a brief period in the sex organs. The potential for PCR-positive tissues from the ovary, uterus, testis and epididymis to transmit infection was investigated via in vitro fertilization (IVF), embryo transfer (ET) or ovary transplantation in immunodeficient athymic nude-nu mice and did not result in contamination of the recipient females.

Tissue distribution and duration of mouse hepatitis virus in naturally infected immunocompetent ICR (CD-1) and immunodeficient athymic nude-nu mouse strains used for ovarian transplantation and in vitro fertilization:

During a natural outbreak of mouse hepatitis virus (MHV) infection in an animal facility in Rome, 6-week-old, outbred, immunocompetent Hsd:ICR (CD-1) and immunodeficient Hsd:athymic nude-nu sentinel mice (barrier maintained) were exposed to MHV in order to study tissue distribution and duration of the virus in naturally infected mice. Infection was diagnosed by serology and by reverse transcriptase-polymerase chain reaction (RT-PCR) using primers directed to two separate but highly conserved regions of the MHV genome. Faeces, colons, spleens, lungs, brains, livers, epididymides, testes, uteri and ovaries from sentinels were tested by RT-PCR after 1, 2, 4, 6, 8 and 12 weeks. A second round of amplification with nested primers was performed to increase the sensitivity of detection. The results indicated that all the organs tested became infected with the virus at various times. Furthermore, male and female reproductive organs were infected within 6 weeks of the beginning of exposure. Investigation of MHV transmission by ovarian transplantation and by in vitro fertilization (IVF) revealed that MHV was transmitted by infected ovaries transplanted into both immunocompetent and immunodeficient mouse strains but transmission was not observed when sperm from infected testes were used for IVF. These results suggest that sperm do not transmit infection from actively infected animals and that IVF could be considered a cleansing procedure.

Contemporary Techniques for Freezing Mouse Spermatozoa

Each year, thousands of new mouse models are generated around the world to further biomedical research. Unfortunately, the cost of maintaining mouse colonies makes it uneconomical to keep strains on the shelf that are not part of active research programs. Ideally, these retired strains should be archived. If this is not done and the line is simply killed off, the genetics are lost to future generations of scientists. Traditionally, embryo freezing has been used to cryopreserve mice, but this is expensive, time consuming, requires large numbers of donor females, and usually involves invasive superovulation procedures. Sperm freezing circumvents all of these disadvantages and is rapidly becoming the technique of choice for many repositories. This has been made possible through the use of refined cryoprotective agents and the development of improved in vitro fertilization techniques. This article describes two popular sperm freezing techniques employed by mouse repositories to archive spermatozoa using cryoprotective agents supplemented with either L‐glutamine or monothioglycerol. Curr. Protoc. Mouse Biol. 4:85‐104

In Vitro Fertilization in Mice Using the MBCD‐GSH Protocol

Historically, timed mating of either naturally cycling or superovulated females has been the mainstay of pre‐implantation embryo production. However, rising cage costs and the rapid expansion of biomedical research programs has necessitated the development of high‐throughput approaches to mouse embryo production. In vitro fertilization (IVF) represents one such versatile tool offering many advantages to busy mouse facilities in terms of efficient use of space and resources. For example, strains can be taken off the shelf, frozen quickly as sperm, and recovered at a later date, small colonies can be rapidly expanded to meet demand, and IVF can be used to rescue strains that fail to breed or where the founder male is ill or has died suddenly. This article describes an IVF protocol currently used by many reproductive technologists to assist mouse biology programs. Curr. Protoc. Mouse Biol. 4:67‐83

Overview of new developments in and the future of cryopreservation in the laboratory mouse

The large-scale mutagenesis programmes underway around the world are generating thousands of novel GA mouse strains that need to be securely archived. In parallel with advances in mutagenesis, the procedures used to cryopreserve mouse stocks are being continually refined in order to keep pace with demand. Moreover, the construction of extensive research infrastructures for systematic phenotyping is fuelling demand for these novel strains of mice and new approaches to the distribution of frozen and unfrozen embryos and gametes are being developed in order to reduce the dependency on the transportation of live mice. This article highlights some contemporary techniques used to archive, rederive, and transport mouse strains around the world.

Transporting Mouse Embryos and Germplasm as Frozen or Unfrozen Materials

The 21st century has seen a huge proliferation in the availability of genetically altered mice. The availability of these resources has been accompanied by ever greater opportunities for international collaborations between laboratories involving the exchange of mouse strains. This exchange can involve significant costs in terms of animal welfare and transportation expenses. In an attempt to mitigate some of these costs, the mouse community has developed a battery of techniques that can be used to avoid transporting live mice. Transporting frozen embryos and sperm at liquid nitrogen (LN2) temperatures using dry shippers has been common practice for some time. However, current advances in this field have refined transportation procedures and introduced new techniques for disseminating embryos and sperm: for example, shipping frozen sperm on dry ice, exchanging unfrozen epididymides from which sperm can be extracted, and transporting frozen/thawed embryos in isotonic media. This article discusses some of the current practices used by laboratories to transport mouse strains around the world without having to exchange live mice. Curr. Protoc. Mouse Biol. 4:47‐65

Blastocyst genotyping for quality control of mouse mutant archives: an ethical and economical approach

With the advent of modern developmental biology and molecular genetics, the scientific community has generated thousands of newly genetically altered strains of laboratory mice with the aim of elucidating gene function. To this end, a large group of Institutions which form the International Mouse Phenotyping Consortium is generating and phenotyping a knockout mouse strain for each of the ~20,000 protein-coding genes using the mutant ES cell resource produced by the International Knockout Mouse Consortium. These strains are made available to the research community via public repositories, mostly as cryopreserved sperm or embryos. To ensure the quality of this frozen resource there is a requirement that for each strain the frozen sperm/embryos are proven able to produce viable mutant progeny, before the live animal resource is removed from cages. Given the current requirement to generate live pups to demonstrate their mutant genotype, this quality control check necessitates the use and generation of many animals and requires considerable time, cage space, technical and economic resources. Here, we describe a simple and efficient method of genotyping pre-implantation stage blastocysts with significant ethical and economic advantages especially beneficial for current and future large-scale mouse mutagenesis projects.

Design and Characterization of a Human Monoclonal Antibody that Modulates Mutant Connexin 26 Hemichannels Implicated in Deafness and Skin Disorders

Background: Mutations leading to changes in properties, regulation, or expression of connexin-made channels have been implicated in 28 distinct human hereditary diseases. Eight of these result from variants of connexin 26 (Cx26), a protein critically involved in cell-cell signaling in the inner ear and skin. Lack of non-toxic drugs with defined mechanisms of action poses a serious obstacle to therapeutic interventions for diseases caused by mutant connexins. In particular, molecules that specifically modulate connexin hemichannel function without affecting gap junction channels are considered of primary importance for the study of connexin hemichannel role in physiological as well as pathological conditions. Monoclonal antibodies developed in the last three decades have become the most important class of therapeutic biologicals. Recombinant methods permit rapid selection and improvement of monoclonal antibodies from libraries with large diversity. Methods: By screening a combinatorial library of human single-chain fragment variable (scFv) antibodies expressed in phage, we identified a candidate that binds an extracellular epitope of Cx26. We characterized antibody action using a variety of biochemical and biophysical assays in HeLa cells, organotypic cultures of mouse cochlea and human keratinocyte-derived cells. Results: We determined that the antibody is a remarkably efficient, non-toxic, and completely reversible inhibitor of hemichannels formed by connexin 26 and does not affect direct cell-cell communication via gap junction channels. Importantly, we also demonstrate that the antibody efficiently inhibits hyperative mutant Cx26 hemichannels implicated in autosomal dominant non-syndromic hearing impairment accompanied by keratitis and hystrix-like ichthyosis-deafness (KID/HID) syndrome. We solved the crystal structure of the antibody, identified residues that are critical for binding and used molecular dynamics to uncover its mechanism of action. Conclusions: Although further studies will be necessary to validate the effect of the antibody in vivo, the methodology described here can be extended to select antibodies against hemichannels composed by other connexin isoforms and, consequently, to target other pathologies associated with hyperactive hemichannels. Our study highlights the potential of this approach and identifies connexins as therapeutic targets addressable by screening phage display libraries expressing human randomized antibodies.

Wnt/β-Catenin Signaling Induces Integrin α4β1 in T Cells and Promotes a Progressive Neuroinflammatory Disease in Mice

The mechanisms leading to autoimmune and inflammatory diseases in the CNS have not been elucidated. The environmental triggers of the aberrant presence of CD4+ T cells in the CNS are not known. In this article, we report that abnormal β-catenin expression in T cells drives a fatal neuroinflammatory disease in mice that is characterized by CNS infiltration of T cells, glial activation, and progressive loss of motor function. We show that enhanced β-catenin expression in T cells leads to aberrant and Th1-biased T cell activation, enhanced expression of integrin α4β1, and infiltration of activated T cells into the spinal cord, without affecting regulatory T cell function. Importantly, expression of β-catenin in mature naive T cells was sufficient to drive integrin α4β1 expression and CNS migration, whereas pharmacologic inhibition of integrin α4β1 reduced the abnormal T cell presence in the CNS of β-catenin–expressing mice. Together, these results implicate deregulation of the Wnt/β-catenin pathway in CNS inflammation and suggest novel therapeutic strategies for neuroinflammatory disorders.