Assessing the welfare of genetically altered mice.

In 2003, under the auspices of the main UK funders of biological and biomedical research, a working group was established with a remit to review potential welfare issues for genetically altered (GA) mice, to summarize current practice, and to recommend contemporary best practice for welfare assessments. The working group has produced a report which makes practical recommendations for GA mouse welfare assessment and dissemination of welfare information between establishments using a 'mouse passport'. The report can be found at www.nc3rs.org.uk/GAmice and www.lal.org.uk/gaa and includes templates for the recommended welfare assessment scheme and the mouse passport. An overview is provided below.

Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs.

Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.

Functional variation of MC1R alleles from red-haired individuals.

Red hair in humans is associated with variant alleles of the alphaMSH receptor gene, MC1R. Loss of MC1R function in other mammals results in red or yellow hair pigmentation. We show that a mouse bacterial artificial chromosome (BAC) which contains Mc1r will efficiently rescue loss of Mc1r in transgenic mice, and that overexpression of the receptor suppresses the effect of the endogenous antagonist, agouti protein. We engineered the BAC to replace the mouse coding region with the human MC1R sequence and used this in the transgenic assay. The human receptor also efficiently rescued Mc1r deficiency, and in addition, appeared to be completely resistant to the effects of agouti, suggesting agouti protein may not play a role in human pigmentary variation. Three human variant alleles account for 60% of all cases of red hair. We engineered each of these in turn into the BAC and find that they have reduced, but not completely absent, function in transgenic mice. Comparison of the phenotypes of alphaMSH-deficient mice and humans in conjunction with this data suggests that red hair may not be the null phenotype of MC1R.

Genetic and environmental factors modify bovine spongiform encephalopathy incubation period in mice.

The incubation period (IP) and the neuropathology of transmissible spongiform encephalopathies (TSEs) have been extensively used to distinguish prion isolates (or strains) inoculated into panels of inbred mouse strains. Such studies have shown that the bovine spongiform encephalopathy (BSE) agent is indistinguishable from the agent causing variant Creutzfeldt-Jakob disease (vCJD), but differs from isolates of sporadic CJD, reinforcing the idea that the vCJD epidemic in Britain results from consumption of contaminated beef products. We present a mouse model for genetic and environmental factors that modify the incubation period of BSE cross-species transmission. We have used two mouse strains that carry the same prion protein (PrP) allele, but display a 100-day difference in their mean IP following intracerebral inoculation with primary BSE isolate. We report genetic effects on IP that map to four chromosomal regions, and in addition we find significant factors of host environment, namely the age of the host's mother, the age of the host at infection, and an X-cytoplasm interaction in the host.

Mouse mutagenesis on target.

Large-scale mutagenesis of the mouse genome is an essential task associated with the Human Genome Project. The two opposing schools of direct and reverse genetics have demonstrated comparable advantages, and yet large numbers of mutant lines have mostly been the prerogative of direct genetics. An improved gene-trapping resource now brings reverse genetics one step closer.

Mouse genomics: making sense of the sequence.

Interpretation of the human genome sequence relies on studies of model genetic organisms. Mouse genetics and genomics will help to identify all the genes, and to determine their function.

Pleiotropic effects of the melanocortin 1 receptor (MC1R) gene on human pigmentation.

Variants of the melanocortin 1 receptor (MC1R) gene are common in individuals with red hair and fair skin, but the relative contribution to these pigmentary traits in heterozygotes, homozygotes and compound heterozygotes for variants at this locus from the multiple alleles present in Caucasian populations is unclear. We have investigated 174 individuals from 11 large kindreds with a preponderance of red hair and an additional 99 unrelated redheads, for MC1R variants and have confirmed that red hair is usually inherited as a recessive characteristic with the R151C, R160W, D294H, R142H, 86insA and 537insC alleles at this locus. The V60L variant, which is common in the population may act as a partially penetrant recessive allele. These individuals plus 167 randomly ascertained Caucasians demonstrate that heterozygotes for two alleles, R151C and 537insC, have a significantly elevated risk of red hair. The shade of red hair frequently differs in heterozygotes from that in homozygotes/compound heterozygotes and there is also evidence for a heterozygote effect on beard hair colour, skin type and freckling. The data provide evidence for a dosage effect of MC1R variants on hair as well as skin colour.

MGF (KIT ligand) is a chemokinetic factor for melanoblast migration into hair follicles.

Melanoblasts, the precursors of the pigment-producing cells of the skin and hair, are derived from the neural crest and migrate to the skin around 12 days of gestation in the mouse. In adult mice almost all the melanoblasts are confined to the hair follicles except for the epidermal layers of nonhairy skin. The receptor tyrosine kinase, KIT, is necessary for the survival, proliferation, and migration of melanoblasts. We have utilised an organ culture for embryonic skin taken from Dct-lacZ transgenic mice. The early patterning of the follicles and developing skin layers is retained within the cultures and the lacZ reporter allows visualisation of the melanoblasts within their native tissue environment. Soon after initiation of hair follicle development, melanoblasts localise in the follicles. Inhibition of follicle formation demonstrates that this localisation is an active process; in the absence of follicles, the melanoblasts proliferate but remain associated with the basement membrane. Implantation of beads releasing MGF, the ligand of KIT, does not result in melanoblast migration towards the bead, rather their localisation to the follicles is accelerated. Addition of soluble MGF induces the same effect; KIT therefore promotes melanocyte movement and acts as a chemokinetic, or motogenic, receptor. The melanoblasts must be guided to their correct location by other chemotactic signals or move at random and locate by ceasing movement when the follicle is engaged.

Melanocortin-1-receptor gene and sun sensitivity in individuals without red hair.

Susceptibility to sunburn, photoageing, and skin cancer is inversely related to an individual's ability to tan after sun exposure. We examined variants in the melanocortin-1-receptor (MC1R) gene in individuals from Ireland and the UK. We found evidence of an association between the degree of tanning after repeated sun exposure, and the number of variant alleles present. Heterozygotes were intermediate between wild-type individuals and those with two variant alleles. We suggest that MC1R gene status therefore determines sun sensitivity in people without red hair.

A late wave of melanoblast differentiation and rostrocaudal migration revealed in patch and rump-white embryos.

Melanocytes originate from a small number of precursors localized either side of the dorsal midline. The tyrosine kinase receptor Kit and its ligand Mgf (Steel Factor) are essential for melanoblast survival and proliferation during their migration from the neural crest. Inappropriate Kit expression in the dermatome and dermis of patch and rump-white mouse mutants apparently sequester Mgf, inhibiting melanoblast dispersal. Using a reporter transgene Dct-lacZ, extensive regions of the mutant trunks appear devoid of melanoblasts between E12.5 and E15.5, a much larger area than seen in mutant adults. Melanoblast recolonization of the underpopulated lumbar regions occurs very rapidly by E16.5 giving rise to patterns consistent with those observed in adults. The mutations permit observation of aspects of melanoblast development that are not seen, or are obscured, in normal embryos.

Evidence for variable selective pressures at MC1R.

It is widely assumed that genes that influence variation in skin and hair pigmentation are under selection. To date, the melanocortin 1 receptor (MC1R) is the only gene identified that explains substantial phenotypic variance in human pigmentation. Here we investigate MC1R polymorphism in several populations, for evidence of selection. We conclude that MC1R is under strong functional constraint in Africa, where any diversion from eumelanin production (black pigmentation) appears to be evolutionarily deleterious. Although many of the MC1R amino acid variants observed in non-African populations do affect MC1R function and contribute to high levels of MC1R diversity in Europeans, we found no evidence, in either the magnitude or the patterns of diversity, for its enhancement by selection; rather, our analyses show that levels of MC1R polymorphism simply reflect neutral expectations under relaxation of strong functional constraint outside Africa.

Melanocortin 1 receptor variation in the domestic dog.

The melanocortin 1 receptor (Mc1r) is encoded by the Extension locus in many different mammals, where a loss-of-function causes exclusive production of red/yellow pheomelanin, and a constitutively activating mutation causes exclusive production of black/brown eumelanin. In the domestic dog, breeds with a wild-type E allele, e. g., the Doberman, can produce either pigment type, whereas breeds with the e allele, e.g., the Golden Retriever, produce exclusively yellow pigment. However, a black coat color in the Newfoundland and similar breeds is thought to be caused by an unusual allele of Agouti, which encodes the physiologic ligand for the Mc1r. Here we report that the predicted dog Mc1r is 317 residues in length and 96% identical to the fox Mc1r. Comparison of the Doberman, Newfoundland, Black Labrador, Yellow Labrador, Flat-coated Retriever, Irish Setter, and Golden Retriever revealed six sequence variants, of which two, S90G and R306ter, partially correlated with a black/brown coat and red/yellow coat, respectively. R306ter was found in the Yellow Labrador, Golden Retriever, and Irish Setter; the latter two had identical haplotypes but differed from the Yellow Labrador at three positions other than R306ter. In a larger survey of 194 dogs and 19 breeds, R306ter and a red/yellow coat were completely concordant except for the Red Chow. These results indicate that the e allele is caused by a common Mc1r loss-of-function mutation that either reoccurred or was subject to gene conversion during recent evolutionary history, and suggest that the allelic and locus relationships for dog coat color genes may be more analogous to those found in other mammals than previously thought.

A comparative transcript map and candidates for mutant phenotypes in the Tyrp1 (brown) deletion complex homologous to human 9p21-23.

The mouse Tyrp1 deletion complex is a valuable resource for high-resolution mapping of genes and phenotypes to the central region of Chromosome (Chr) 4. The distal part of the complex is homologous to human Chr 9p21-23, and we have used the available radiation hybrid maps to identify human transcripts in the region. We localize seven genes to a human YAC contig that spans the full extent of the distal deletion complex and show that the mouse homologs of four of these, including Cer1, map within the complex. On the basis of location and/or expression, we exclude genes as candidates for several known phenotypes in the region and identify a candidate transcript for the neonatal lethal phenotype l(4)Rn2.

The mahogany mouse mutation: further links between pigmentation, obesity and the immune system.

Completely different lines of experimentation have identified attractin, a protein that seems to have multiple roles in regulating physiological processes. It affects the balance between agonist and antagonist at receptors on melanocytes, modifies behaviour and basal metabolic rate, and mediates an interaction between activated T cells and macrophages. It may well be a target for development of drugs to treat obesity.

Deficiency of Trp53 rescues the male fertility defects of Kit(W-v) mice but has no effect on the survival of melanocytes and mast cells.

Mutations of the receptor tyrosine kinase, Kit, or its ligand, mast growth factor (Mgf), affect three unrelated cell populations: melanocytes, germ cells, and mast cells. Kit signaling is required initially to prevent cell death in these lineages both in vitro and in vivo. Mgf appears to play a role in the survival of some hematopoietic cells in vitro by modulating the activity of p53. Signaling by Mgf inhibits p53-induced apoptosis of erythroleukemia cell lines and suppresses p53-dependent radiation-induced apoptosis of bone marrow cells. We tested the hypothesis that cell survival in Kit mutant mice would be enhanced by p53 deficiency in vivo. Double-mutant mice, which have greatly reduced Kit receptor tyrosine kinase activity and also lack Trp53, were generated and the affected cell lineages examined. Mast cell, melanoblast, and melanocyte survival in the double Kit(W-v/W-v):Trp53(-/-) mutants was not increased compared to the single Kit(W-v/W-v):Trp53(+/+) mutants. However, double-mutant males showed an increase in sperm viability and could father litters, in contrast to their homozygous Kit mutant, wild-type p53 littermates. This germ cell rescue appears to be male specific, as female ovaries were similar in mice homozygous for the Kit mutant allele with or without p53. We conclude that defective Kit signaling in vivo results in apoptosis by a p53-independent pathway in melanocyte and mast cell lineages but that in male germ cells apoptosis in the absence of Kit is p53-dependent.

The mouse Cer1 (Cerberus related or homologue) gene is not required for anterior pattern formation.

Cer1 is the mouse homologue of the Xenopus Cerberus gene whose product is able to induce development of head structures during embryonic development. The Cer1 protein is a member of the cysteine knot superfamily and is expressed in anterior regions of the mouse gastrula. A segmental pattern of expression with nascent and newly formed somites is also seen. This suggests an additional role in development of the axial skeleton, musculature, or peripheral nervous system. Xenopus animal cap assays and mouse germ-layer explant recombination experiments indicate that the mouse protein can act as a patterning molecule for anterior development in Xenopus, including induction of Otx2 expression, and suggest it may have a similar role in mouse development. However, we present here genetic data that demonstrate that Cer1 is not necessary for anterior patterning, Otx2 expression, somite formation, or even normal mouse morphogenesis.

Identification, sequence, and mapping of the mouse multiple PDZ domain protein gene, Mpdz.

The PDZ domain gained its name from the three proteins that were first seen to have homology by virtue of these domains, the mammalian postsynaptic density protein, PSD-95, the Drosophila discs-large septate junction protein, DLG, and the mammalian epithelial tight-junction protein zona occludens, ZO-1. Over 50 PDZ domain-containing genes have been recognized so far from almost any organism subjected to sequencing, including mammals, nematodes, yeast, plants, and bacteria. The domain consists of an approximately 90-amino-acid-residue unit, which is often repeated in the protein. The majority of residues form a conserved spatial structure while a few amino acids in critical positions confer protein binding specificity. A subgroup of PDZ domains have been shown to recognize a short carboxy-terminal amino acid motif, T/SXV (Ser/Thr-X-Val-COO-), where X is any amino acid. We have identified and completely sequenced a gene, Mpdz, that encodes a mouse protein containing 13 such domains. We have also mapped the gene to a series of overlapping deletions on mouse chromosome 4 and can therefore determine that its function is not essential for embryonic development or neonatal survival.

Melanocortin receptors and antagonists regulate pigmentation and body weight.

The action two genetic loci--agouti and the melanocortin receptor-1 (Mc1r)-- have opposing effects in the control of mammalian pigmentation and ultimately determine the color of the pigment produced. In a recent paper, Ollmann et al. confirmed that the agouti protein acts via the Mc1r. They show that high-affinity binding of the agouti protein to Mc1r expressed in mammalian cells can be inhibited by the receptor's natural ligand, alpha-melanocyte-stimulating hormone (alpha-MSH). In addition, genetic studies using mice carrying mutations at the Mc1r and agouti loci on a sensitized background of low tyrosinase expression confirm that a functional Mc1r is required for the maximum pigmentary effect of agouti. Thus, the Mc1r appears to be a unique, bifunctionally controlled receptor, activated by alpha-MSH and antagonized by agouti, both of which contribute to the variability seen in mammalian coat color.