Sigma (sigma) sites are a type of nonopiate receptor whose role has been associated with several behaviours, including anxiety, depression, analgesia, learning processes and psychosis. Although there are several known sigma receptor types, only the type I receptor (sigma 1) has been cloned. To uncover the in vivo relevance of sigma-receptors, we have generated knockout mice for sigma 1. Despite the broad expression pattern found for the sigma 1-gene, homozygous mutant mice are viable, fertile and do not display any overt phenotype, compared with their wild-type litter-mates, in mixed genetic backgrounds. However, a significant decrease in the hypermotility response has been measured in knockout mice upon challenge with (+)SKF-10 047, in agreement with the involvement of sigma 1-receptors in the induction of psychostimulant actions. The activity of sigma 2-receptors seems to be unaffected in sigma 1-mutant mice. These knockout mice could contribute to better understand the in vivo role of sigma-receptors.

Mice from the inbred C57BL/6 strain have been commonly used for the generation and analysis of transgenic and knockout animal models. However, several C57BL/6 substrains exist, and these are genetically and phenotypically different. In addition, each of these substrains can be purchased from different animal providers and, in some cases, they have maintained their breeding stocks separated for a long time, allowing genetic differences to accumulate due to individual variability and genetic drift. With the aim of describing the differences in the genotype of several C57BL/6 substrains, we applied the Illumina(®) Mouse Medium Density Linkage Mapping panel, with 1,449 single nucleotide polymorphisms (SNPs), to individuals from ten C57BL/6-related strains: C57BL/6JArc, C57BL/6J from The Jackson Lab, C57BL/6J from Crl, C57BL6/JRccHsd, C57BL/6JOlaHsd, C57BL/6JBomTac, B6(Cg)-Tyr ( c-2j )/J, C57BL/6NCrl, C57BL/6NHsd and C57BL/6NTac. Twelve SNPs were found informative to discriminate among the mouse strains considered. Mice derived from the original C57BL/6J: C57BL/6JArc, C57BL/6J from The Jackson Lab and C57BL/6J from Crl, were indistinguishable. Similarly, all C57BL/6N substrains displayed the same genotype, whereas the additional substrains showed intermediate cases with substrain-specific polymorphisms. These results will be instrumental for the correct genetic monitoring and appropriate mouse colony handling of different transgenic and knockout mice produced in distinct C57BL/6 inbred substrains.

Newly developed genome-editing tools, such as the clustered regularly interspaced short palindromic repeat (CRISPR)–Cas9 system, allow simple and rapid genetic modification in most model organisms and human cell lines. Here, we report the production and analysis of mice carrying the inactivation via deletion of a genomic insulator, a key non-coding regulatory DNA element found 5′ upstream of the mouse tyrosinase (Tyr) gene. Targeting sequences flanking this boundary in mouse fertilized eggs resulted in the efficient deletion or inversion of large intervening DNA fragments delineated by the RNA guides. The resulting genome-edited mice showed a dramatic decrease in Tyr gene expression as inferred from the evident decrease of coat pigmentation, thus supporting the functionality of this boundary sequence in vivo, at the endogenous locus. Several potential off-targets bearing sequence similarity with each of the two RNA guides used were analyzed and found to be largely intact. This study reports how non-coding DNA elements, even if located in repeat-rich genomic sequences, can be efficiently and functionally evaluated in vivo and, furthermore, it illustrates how the regulatory elements described by the ENCODE and EPIGENOME projects, in the mouse and human genomes, can be systematically validated.

SummaryIn animal cells, microtubule and actin tracks and their associated motors (dynein, kinesin, and myosin) are thought to regulate long- and short-range transport, respectively [1–8]. Consistent with this, microtubules extend from the perinuclear centrosome to the plasma membrane and allow bidirectional cargo transport over long distances (>1 μm). In contrast, actin often comprises a complex network of short randomly oriented filaments, suggesting that myosin motors move cargo short distances. These observations underpin the “highways and local roads” model for transport along microtubule and actin tracks [2]. The “cooperative capture” model exemplifies this view and suggests that melanosome distribution in melanocyte dendrites is maintained by long-range transport on microtubules followed by actin/myosin-Va-dependent tethering [5, 9]. In this study, we used cell normalization technology to quantitatively examine the contribution of microtubules and actin/myosin-Va to organelle distribution in melanocytes. Surprisingly, our results indicate that microtubules are essential for centripetal, but not centrifugal, transport. Instead, we find that microtubules retard a centrifugal transport process that is dependent on myosin-Va and a population of dynamic F-actin. Functional analysis of mutant proteins indicates that myosin-Va works as a transporter dispersing melanosomes along actin tracks whose +/barbed ends are oriented toward the plasma membrane. Overall, our data highlight the role of myosin-Va and actin in transport, and not tethering, and suggest a new model in which organelle distribution is determined by the balance between microtubule-dependent centripetal and myosin-Va/actin-dependent centrifugal transport. These observations appear to be consistent with evidence coming from other systems showing that actin/myosin networks can drive long-distance organelle transport and positioning [10, 11].

SummaryStrial melanocytes are required for normal development and correct functioning of the cochlea. Hearing deficits have been reported in albino individuals from different species, although melanin appears to be not essential for normal auditory function. We have analyzed the auditory brainstem responses (ABR) of two transgenic mice: YRT2, carrying the entire mouse tyrosinase (Tyr) gene expression-domain and undistinguishable from wild-type pigmented animals; and TyrTH, non-pigmented but ectopically expressing tyrosine hydroxylase (Th) in melanocytes, which generate the precursor metabolite, L-DOPA, but not melanin. We show that young albino mice present a higher prevalence of profound sensorineural deafness and a poorer recovery of auditory thresholds after noise-exposure than transgenic mice. Hearing loss was associated with absence of cochlear melanin or its precursor metabolites and latencies of the central auditory pathway were unaltered. In summary, albino mice show impaired hearing responses during ageing and after noise damage when compared to YRT2 and TyrTH transgenic mice, which do not show the albino-associated ABR alterations. These results demonstrate that melanin precursors, such as L-DOPA, have a protective role in the mammalian cochlea in age-related and noise-induced hearing loss. SignificanceThis manuscript describes how melanin precursors, such as L-DOPA, prevent the profound premature age-related deafness and noise-induced hearing loss associated with albinism in mice. We use two welldefined transgenic mouse models of oculocutaneous albinism type I to study the auditory deficits associated with albinism in mice. We show that melanin and L-DOPA can prevent these hearing alterations. Since L-DOPA alone, a melanin precursor, is enough to rescue the observed deficits we conclude that melanin precursors produced by melanocytes is all what is needed to restore the alterations observed in albino mice by ABR. We do not know the exact mechanism nor we have determined the cause of the hearing loss in these albino mice. However, since melanin (and L-DOPA) can bind calcium we propose that the observed hearing loss phenotype might be caused by alterations in the calcium homeostasis of the endolymph, produced by the stria vascularis of the cochlea, where melanocytes are located within the inner ear. Our work might also be relevant for the corresponding genetic disorder in humans, where, to date, no systematic studies regarding auditory function have been carried out.

Cell biologists generally consider that microtubules and actin play complementary roles in long-and short-distance transport in animal cells. On the contrary, using melanosomes of melanocytes as a model, we recently discovered that the motor protein myosin-Va works with dynamic actin tracks to drive long-range organelle dispersion in opposition to microtubules. This suggests that in animals, as in yeast and plants, myosin/actin can drive longrange transport. Here, we show that the SPIRE-type actin nucleators (predominantly SPIRE1) are Rab27a effectors that cooperate with formin-1 to generate actin tracks required for myosin-Va-dependent transport in melanocytes. Thus, in addition to melanophilin/myosin-Va, Rab27a can recruit SPIREs to melanosomes, thereby integrating motor and track assembly activity at the organelle membrane. Based on this, we suggest a model in which organelles and force generators (motors and track assemblers) are linked, forming an organelle-based, cell-wide network that allows their collective activity to rapidly disperse the population of organelles long-distance throughout the cytoplasm.

This 'highways and local roads' model suggests that MTs are tracks for long-range transport (highways) between the cell centre and periphery, driven by kinesin and dynein motors. Meanwhile AFs (local roads) and myosin motors work down-stream picking up cargo at the periphery and transporting it for the 'last m' to its final destination. This model makes intuitive sense as MTs in animal cells in culture typically form a polarised radial network of tracks spanning >10 m from the centrally located centrosome to the periphery and appear ideally distributed for long-distance transport. Meanwhile, with some exceptions in which AFs form uniformly polarised arrays, e.g. lamellipodia, filopodia and dendritic spines, AF architecture appears much more complex. In many fixed cells AF appear to comprise populations of short (1-2 m length), with random or anti-parallel filament polarity, and not an obvious system of tracks for directed transport 5,6 . This view is exemplified by the co-operative capture (CC) model of melanosome transport in melanocytes 7,8 . Skin melanocytes make pigmented melanosomes and then distribute them, via dendrites, to adjacent keratinocytes, thus providing pigmentation and photo-protection (reviewed in 9 ). The CC model proposes that transport of melanosomes into dendrites occurs by sequential longdistance transport from the cell body into dendrites along MTs (propelled by kinesin/dynein motors), followed by AF/myosin-Va dependent tethering in the dendrites. Consistent with this, in myosin-Va-null cells melanosomes move bi-directionally along MTs into dendrites, but do not accumulate therein, and instead cluster in the cell body 7,10 . This defect results in partial albinism in mammals due to uneven pigment transfer from melanocytes to keratinocytes (e.g. dilute mutant mouse and human Griscelli syndrome (GS) type I patients; Figure 1A) 11,12 . Subsequent studies revealed similar defects in mutant mice (and human GS types II and III patients) lacking the small

The simple protocol described in this article aims to provide all required information, as a comprehensive, easy‐to‐follow step‐by‐step method, to ensure the generation of the expected genome‐edited mice. Here, we provide protocols for the preparation of CRISPR‐Cas9 reagents for microinjection and electroporation into one‐cell mouse embryos to create knockout or knock‐in mouse models, and for genotyping the resulting offspring with the latest innovative next‐generation sequencing methods. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Designing the best RNA guide for your gene disruption/editing strategy Basic Protocol 2: Preparing and validating CRISPR‐Cas9 reagents Basic Protocol 3: Preparing and injecting CRISPR‐Cas9 compounds into fertilized mouse oocytes Basic Protocol 4: Genotyping genome‐edited mice Support Protocol: Genotyping for CRISPR‐generated “indel” mutations