Response of melanocortin-4 receptor-deficient mice to anorectic and orexigenic peptides.

Mutations reducing the functional activity of leptin, the leptin receptor, alpha-melanocyte stimulating hormones (alpha-MSH) and the melanocortin-4 receptor (Mc4r) all lead to obesity in mammals. Moreover, mutant mice that ectopically express either agouti (Ay/a mice) or agouti-related protein (Agrp), antagonists of melanocortin signalling, become obese. These data suggest that alpha-MSH signalling transduced by Mc4r tonically inhibits feeding; however, it is not known to what extent this pathway mediates leptin signalling. We show here that Mc4r-deficient (Mc4r-/-) mice do not respond to the anorectic actions of MTII, an MSH-like agonist, suggesting that alpha-MSH inhibits feeding primarily by activating Mc4r. Obese Mc4r-/-mice do not respond significantly to the inhibitory effects of leptin on feeding, whereas non-obese Mc4r-/- mice do. These data demonstrate that melanocortin signalling transduced by Mc4r is not an exclusive target of leptin action and that factors resulting from obesity contribute to leptin resistance. Leptin resistance of obese Mc4r-/- mice does not prevent their response to the anorectic actions of ciliary neurotrophic factor (CNTF), corticotropin releasing factor (CRF), or urocortin; or the orexigenic actions of neuropeptide Y (NPY) or peptide YY (PYY), indicating that these neuromodulators act independently or downstream of Mc4r signalling.

A role for smad6 in development and homeostasis of the cardiovascular system.

Smad proteins are intracellular mediators of signalling initiated by Tgf-betasuperfamily ligands (Tgf-betas, activins and bone morphogenetic proteins (Bmps)). Smads 1, 2, 3, 5 and 8 are activated upon phosphorylation by specific type I receptors, and associate with the common partner Smad4 to trigger transcriptional responses. The inhibitory Smads (6 and 7) are transcriptionally induced in cultured cells treated with Tgf-beta superfamily ligands, and downregulate signalling in in vitro assays. Gene disruption in mice has begun to reveal specific developmental and physiological functions of the signal-transducing Smads. Here we explore the role of an inhibitory Smad in vivo by targeted mutation of Madh6 (which encodes the Smad6 protein). Targeted insertion of a LacZ reporter demonstrated that Smad6 expression is largely restricted to the heart and blood vessels, and that Madh6 mutants have multiple cardiovascular abnormalities. Hyperplasia of the cardiac valves and outflow tract septation defects indicate a function for Smad6 in the regulation of endocardial cushion transformation. The role of Smad6 in the homeostasis of the adult cardiovascular system is indicated by the development of aortic ossification and elevated blood pressure in viable mutants. These defects highlight the importance of Smad6 in the tissue-specific modulation of Tgf-beta superfamily signalling pathways in vivo.

Phase I/II multicenter study to assess the safety, tolerability, pharmacokinetics and pharmacodynamics of AZD4877 in patients with refractory acute myeloid leukemia.

Eg5 (kinesin spindle protein) is a microtubule motor protein, essential for centrosome separation during mitosis. This Phase I/II, open-label, multicenter, two-part study investigated AZD4877, a potent Eg5 inhibitor, in patients with acute myeloid leukemia. Primary objectives were to determine the maximum tolerated dose (MTD) (part A), assess efficacy (part B) and determine the pharmacokinetic profile (parts A and B). Secondary objectives included assessment of safety and tolerability. AZD4877 was administered at a range of doses (2, 4, 7, 10, 13, 16 and 18 mg/day) as a 1-hour intravenous infusion on three consecutive days of a continuous 2-week schedule. The MTD in part A was defined as 16 mg/day based on dose-limiting stomatitis at 16 and 18 mg/day, hyperbilirubinemia at 16 mg/day and palmar-plantar erythrodysesthesia syndrome at 18 mg/day. Systemic exposure to AZD4877 generally increased with increasing dose whereas half-life was not dose dependent. No evaluable patients experienced a complete remission (CR) or CR with incomplete blood count recovery (CRi), demonstrating no evidence of AZD4877 efficacy in this population. Evidence of monoasters in all but the 4 mg/day dose group provided proof of mechanism for AZD4877. This study was terminated due to lack of efficacy. (ClinicalTrials.gov identifier NCT00486265).

Light chain editing in kappa-deficient animals: a potential mechanism of B cell tolerance.

The genetic organization of the kappa and lambda light chain loci permits multiple, successive rearrangement attempts at each allele. Multiple rearrangements allow autoreactive B cells to escape clonal deletion by editing their surface receptors. Editing may also facilitate efficient B cell production by salvaging cells with nonproductive light chain (L chain) rearrangements. To study receptor editing of kappa L chains, we have characterized B cells from mice hemizygous for the targeted inactivation of kappa (JCkD/wt) which have an anti-DNA heavy chain transgene, 3H9. Hybridomas from JCkD/wt mice exhibited an increased frequency of rearrangements to downstream Jk segments (such as Jk5) compared with most surveys from normal mice, consistent with receptor editing by sequential kappa locus rearrangements in JCkD/wt. We observed an even higher frequency of rearrangements to Jk5 in 3H9 JCkD/wt animals compared with nontransgenic JCkD/wt, consistent with editing of autoreactive kappa in 3H9 JCkD/wt. We also recovered a large number of 3H9 JCkD/wt lines with Vk12/13-Jk5 rearrangements and could demonstrate by PCR and Southern analysis that up to three quarters of these lines underwent multiple kappa rearrangements. To investigate editing at the lambda locus, we used homozygous kappa-deficient animals (JCkD/JCkD and 3H9 JCkD/JCkD). The frequencies of V lambda 1 and V lambda 2 rearrangements among splenic hybridomas in 3H9 JCkD/JCkD were reduced by 75% whereas V lambda X was increased 5-10-fold, compared with nontransgenic JCkD/JCkD animals. This indicates that V lambda 1 and V lambda 2 are negatively regulated in 3H9 JCkD/JCkD, consistent with earlier studies that showed that the 3H9 heavy chain, in combination with lambda 1 binds DNA. As successive lambda rearrangements to V lambda X do not inactivate V lambda 1, the consequence of lambda editing in 3H9 JCkD/JCkD would be failed allelic exclusion at lambda. However, analysis of 18 3H9 JCkD/JCkD hybridomas with V lambda 1 and V lambda X DNA rearrangements revealed that most of these lines do not have productive lambda 1 rearrangements. In sum, both kappa and lambda loci undergo editing to recover from nonproductive rearrangement, but only kappa locus editing appears to play a substantial role in rescuing autoreactive B cells from deletion.

The role of B cells in lpr/lpr-induced autoimmunity.

The primary roles of T cells and B cells in the initiation of systemic autoimmunity are unclear. To investigate the role of B cells, we crossed the "Jh knockout" mutation onto the autoimmune lpr/lpr background. Animals homozygous for both traits were obtained. As expected, these animals lack B cells. These animals also show no signs of autoimmune kidney destruction nor vasculitis, in spite of carrying the lpr/lpr mutation. In contrast, lpr/lpr littermates that had B cells had severe nephritis and vasculitis, as well as autoantibodies. These results demonstrate a primary role for B cells and/or (auto)antibodies in initiating several types of autoimmune-mediated tissue destruction. The implications of this finding for models and therapy of autoimmunity are discussed.

Targeted deletion of the tub mouse obesity gene reveals that tubby is a loss-of-function mutation.

The mouse tubby phenotype is characterized by maturity-onset obesity accompanied by retinal and cochlear degeneration. A positional cloning effort to find the gene responsible for this phenotype led to the identification of tub, a member of a novel gene family of unknown function. A splice defect mutation in the 3' end of the tub gene, predicted to disrupt the C terminus of the Tub protein, has been implicated in the genesis of the tubby phenotype. It is not clear, however, whether the Tub mutant protein retains any biological activity, or perhaps has some dominant function, nor is it established that the tubby mutation is itself responsible for all of the observed tubby phenotypes. To address these questions, we generated tub-deficient mice and compared their phenotype to that of tubby mice. Our results demonstrate that tubby is a loss-of-function mutation of the tub gene and that loss of the tub gene is sufficient to give rise to the full spectrum of tubby phenotypes. We also demonstrate that loss of photoreceptors in the retina of tubby and tub-deficient mice occurs by apoptosis. In addition, we show that Tub protein expression is not significantly altered in the ob, db, or melanocortin 4 receptor-deficient mouse model of obesity.

Induction of neuropeptide Y gene expression in the dorsal medial hypothalamic nucleus in two models of the agouti obesity syndrome.

Dominant mutations at the agouti locus induce several phenotypic changes in the mouse including yellow pigmentation (phaeomelanization) of the coat and adult-onset obesity. Nonpigmentary phenotypic changes associated with the agouti locus are due to ectopic expression of the agouti-signaling protein (ASP), and the pheomelanizing effects on coat color are due to ASP antagonism of alpha-MSH binding to the melanocyte MC1 receptor. Recently it has been demonstrated that pharmacological antagonism of hypothalamic melanocortin receptors or genetic deletion of the melanocortin 4 receptor (MC4-R) recapitulates aspects of the agouti obesity syndrome, thus establishing that chronic disruption of central melanocortinergic signaling is the cause of agouti-induced obesity. To learn more about potential downstream effectors involved in these melanocortinergic obesity syndromes, we have examined expression of the orexigenic peptides galanin and neuropeptide Y (NPY), as well as the anorexigenic POMC in lethal yellow (A(y)), MC4-R knockout (MC4-RKO), and leptin-deficient (ob/ob) mice. No significant changes in galanin or POMC gene expression were seen in any of the obese models. In situ hybridizations using an antisense NPY probe demonstrated that in obese A(y) mice, arcuate nucleus NPY mRNA levels were equivalent to that of their C57BL/6J littermates. However, NPY was expressed at high levels in a new site, the dorsal medial hypothalamic nucleus (DMH). Expression of NPY in the DMH was also seen in obese MC4-RKO homozygous (-/-) mice, but not in lean heterozygous (+/-) or wild type (+/+) control mice. This identifies the DMH as a brain region that is functionally altered by the disruption of melanocortinergic signaling and suggests that this nucleus, possibly via elevated NPY expression, may have an etiological role in the melanocortinergic obesity syndrome.

High efficiency gene transfer and expression in normal murine B lymphocytes.

The introduction of new genetic information into hematopoietic cells offers a new approach for investigating the molecular events controlling differentiation. Retrovirus vectors have been used to transfer new genes with high efficiency into murine hematopoietic cells, primarily of the myeloid lineage. In this report, we show that vectors carrying the dominant, selectable gene for neomycin resistance (neo gene) can successfully infect normal murine B lymphocytes (CFU-B). The infected CFU-B formed colonies in vitro in high concentrations (750 micrograms/ml) of G418, a neomycin analogue. That B lymphocytes contained the neo gene was confirmed by the findings that the putative B cell colonies growing in G418 contained antibody-producing cells and that the cells responding to the B cell mitogen, LPS, were resistant to G418. Infection of normal spleen cells with different vectors containing a variety of transcriptional regulatory sequences resulted in 7-40% of the CFU-B becoming G418 resistant. Introduction of the immunoglobulin heavy chain enhancer into NEO vectors appeared to augment the expression of the neo gene, since the level of G418 resistance was higher in B cells infected with a NEO vector containing the enhancer than in cells infected with a vector lacking the enhancer.

Establishment and chimera analysis of 129/SvEv- and C57BL/6-derived mouse embryonic stem cell lines.

Hundreds of new mutant mouse lines are being produced annually using gene targeting and gene trap approaches in embryonic stem (ES) cells, and the number is expected to continue to grow as the human and mouse genome projects progress. The availability of robust ES cell lines and a simple technology for making chimeras is more attractive now than ever before. We established several new ES cell lines from 129/SvEv and C57BL/6 mice and tested their ability to contribute to the germline following blastocyst injections and/or the less expensive and easier method of morula-ES cell aggregation. Using morula aggregation to produce chimeras, five newly derived 129/SvEv and two C57BL/6 ES cell lines tested at early passages were found to contribute extensively to chimeras and produce germline-transmitting male chimeras. Furthermore, the two 129S/vEv ES cell lines that were tested and one of the C57BL/6 ES cell lines were able to maintain these characteristics after many passages in vitro. Our results indicate that the ability of ES cells to contribute strongly to chimeras following aggregation with outbred embryos is a general property of early passage ES cells and can be maintained for many passages. C56BL/6-derived ES cell lines, however, have a greater tendency than 129-derived ES cell lines to lose their ability to colonize the germline.

Human antibodies from transgenic mice.

We have used homologous recombination in ES cells to engineer B cell-deficient mice that are incapable of expressing endogenous immunoglobulin heavy and kappa light chain genes. We find that B cell development in these mutant mice can be rescued by the introduction of human germline-configuration heavy- and kappa light-chain minilocus transgenes. The transgenes rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation in response to antigen stimulation; thus recapitulating both stages of the humoral immune response using human, rather than mouse, sequences. The mice can be immunized; and human sequence, antigen specific, monoclonal antibodies can be obtained using conventional rodent hybridoma technology. These animals are also of interest for studying the normal processes of immunoglobulin gene expression. We discuss the example of heavy chain class switching, which has not been previously observed within an autonomous transgene.

A Phase I study to assess the safety, tolerability, and pharmacokinetics of AZD4877, an intravenous Eg5 inhibitor in patients with advanced solid tumors.

PURPOSE: Inhibition of kinesin spindle protein or Eg5 causes the formation of monoastral mitotic spindles, which leads to cell death. AZD4877 is a specific, potent inhibitor of Eg5. METHODS: This was a Phase I, open-label, two-part study to evaluate the maximum tolerated dose (MTD) and safety and tolerability of AZD4877 in patients with advanced solid malignancies. In part A, the MTD of AZD4877, administered as three weekly 1-h intravenous (iv) infusions in a 28-day schedule, was determined by evaluating dose-limiting toxicity (DLT). In part B, the safety, tolerability, and pharmacokinetic profile of AZD4877 at the MTD were evaluated. RESULTS: In part A, 29 patients received at least one dose of AZD4877 (5 mg, n = 4; 7.5 mg, n = 4; 10 mg, n = 3; 15 mg, n = 3; 20 mg, n = 3; 30 mg, n = 6; 36 mg, n = 3; 45 mg, n = 3). The MTD was defined as 30 mg, with the primary DLT being neutropenia. Although exposures appeared to be similar at the AZD4877 20 and 30 mg doses, dose reductions and omissions were higher in the 30-mg cohort; therefore, an intermediate dose, 25 mg, was evaluated in part B (n = 14). In part B, neutropenia remained the most commonly reported causally related adverse event. Exposure to AZD4877 was approximately dose proportional. Severity of neutropenia was related to exposure. CONCLUSION: The MTD of AZD4877 given as a 1-h iv infusion on days 1, 8, and 15 of a 28-day cycle was 30 mg. At the selected 25 mg dose, AZD4877 had an acceptable safety profile.

Is ribonucleotide reductase the transforming function of herpes simplex virus 2?

Transformation of cells by herpes simplex virus 2 (HSV-2) can be induced by the BglII C (0.43-0.58 map units) or N (0.58-0.625) fragments of the viral genome. Sequences partially overlapping both fragments (0.566-0.602) encode two 3' coterminal mRNAs; these in turn direct the synthesis of two related polypeptides of molecular weight 140,000 (140K) and 35K (refs 4, 7), which may be involved in transformation. Recently, a temperature-sensitive (ts) mutation affecting HSV-induced ribonucleotide reductase has been mapped within this common region (B.M. Dutia, personal communication). We have partially purified the induced reductase and raised a rabbit antiserum to it which inhibits the enzyme activity and immunoprecipitates from infected cells a 144K polypeptide and minor species including a 38K polypeptide. Here we show that a monoclonal antibody to the putative transforming proteins competes with the rabbit serum for the 144K and 38K antigens and also immunoprecipitates specifically the induced reductase activity. These results suggest a possible role for ribonucleotide reductase in HSV-2-induced transformation.

Partial purification and characterization of the ribonucleotide reductase induced by herpes simplex virus infection of mammalian cells.

In this report we confirm and further characterize the induction of a novel ribonucleotide reductase after herpes simplex virus infection of mammalian cells. Induction of the enzyme was observed at a multiplicity of infection of 1 PFU/cell or greater and was found to be maximal (three- to sixfold the activity in mock-infected controls at 6 to 8 h postinfection at a multiplicity of infection of 10 PFU/cell. Partial purification and subsequent characterization of the reductase activity from infected cells demonstrated the existence of two enzymes which could be separated by precipitation with ammonium sulfate. One of the activities precipitated at between 35 and 55% salt saturation, as did the enzyme from control cells, whereas the novel activity precipitated at 0 to 35% saturation. This latter enzyme was similar to the herpes simplex virus-induced reductase described by others in its lack of requirement for Mg2 and its resistance to inhibition by dTTP and dATP; in addition, we found that it was inhibited by ATP, whereas the enzyme from control cells displayed an absolute requirement for the nucleotide. Both enzymes were equally inhibited by pyridoxal phosphate and showed similar cold and heat stability. The enzyme induced by herpes simplex virus infection, however, was much more labile than the control enzyme upon purification.

Migration and proliferation of cultured neural crest cells in W mutant neural crest chimeras.

Chimeric mice, generated by aggregating preimplantation embryos, have been instrumental in the study of the development of coat color patterns in mammals. This approach, however, does not allow for direct experimental manipulation of the neural crest cells, which are the precursors of melanoblasts. We have devised a system that allows assessment of the developmental potential and migration of neural crest cells in vivo following their experimental manipulation in vitro. Cultured C57Bl/6 neural crest cells were microinjected in utero into neurulating Balb/c or W embryos and shown to contribute efficiently to pigmentation in the host animal. The resulting neural crest chimeras showed, however, different coat pigmentation patterns depending on the genotype of the host embryo. Whereas Balb/c neural crest chimeras showed very limited donor cell pigment contribution, restricted largely to the head, W mutant chimeras displayed extensive pigmentation throughout, often exceeding 50% of the coat. In contrast to Balb/c chimeras, where the donor melanoblasts appeared to have migrated primarily in the characteristic dorsoventral direction, in W mutants the injected cells appeared to migrate in the longitudinal as well as the dorsoventral direction, as if the cells were spreading through an empty space. This is consistent with the absence of a functional endogenous melanoblast population in W mutants, in contrast to Balb/c mice, which contain a full complement of melanocytes. Our results suggest that the W mutation disturbs migration and/or proliferation of endogenous melanoblasts. In order to obtain information on clonal size and extent of intermingling of donor cells, two genetically marked neural crest cell populations were mixed and coinjected into W embryos. In half of the tricolored chimeras, no co-localization of donor crest cells was observed, while, in the other half, a fine intermingling of donor-derived colors had occurred. These results are consistent with the hypothesis that pigmented areas in the chimeras can be derived from extensive proliferation of a few donor clones, which were able to colonize large territories in the host embryo. We have also analyzed the development of pigmentation in neural crest cultures in vitro, and found that neural tubes explanted from embryos carrying wt or weak W alleles produced pigmented melanocytes while more severe W genotypes were associated with deficient pigment formation in vitro.

Herpes simplex virus-induced ribonucleotide reductase: development of antibodies specific for the enzyme.

We have previously reported on the characterization of a novel ribonucleotide reductase induced in herpes simplex virus type 2 (HSV-2)-infected mammalian cells. The virus-induced enzyme was partially purified, free of the constitutive cell isozyme, by fractionation of infected cell extracts with ammonium sulphate. In this report we describe a further purification of the virus-induced enzyme and the development of a rabbit antiserum capable of specifically inhibiting its activity. Enzymically active salt fractions from infected cell extracts were sedimented through glycerol gradients; the virus-induced enzyme was found to sediment approximately 2 . 5 times faster than the constitutive, or control, enzyme and was separated from the majority of the protein in the sample. Immunization of rabbits with the partially purified enzyme preparation recovered from gradients resulted in the synthesis of antibodies which completely and specifically inhibited the virus-induced reductase, and precipitated one major polypeptide and a few minor species from both HSV-1- and HSV-2-infected cells. The antibodies, however, exhibited much greater affinity for the HSV-2 than the HSV-1 antigen. These results demonstrate that the virus-induced enzyme differs antigenically, as well as biochemically, from the constitutive cell isozyme and lend further support to the hypothesis that the enzyme is virus-coded.

Effects of mutations at the W locus (c-kit) on inner ear pigmentation and function in the mouse.

The W locus encodes a tyrosine kinase receptor, c-kit, which affects survival of melanoblasts from the neural crest. The primary cochlear defect in Viable Dominant Spotting (Wv/Wv) mutants is a lack of melanocytes within the stria vascularis (SV) associated with an endocochlear potential (EP) close to zero and hearing impairment. In this study, we compare inner ear pigmentation with cochlear potentials in three other W alleles (Wx, Wsh, and W41) and reveal an unequivocal correlation between presence of strial melanocytes and presence of an EP. Asymmetry was common, and 8.3% of Wsh/Wx, 25% of Wsh/Wsh, 60% of W41/Wx, and 69.2% of W41/W41 ears had a pigmented stria and an EP, while the remainder had no strial melanocytes and no EP. In those mutants that partially escaped the effects of the mutation, strial melanocytes rarely extended the entire length of the stria, but were confined to the middle and/or basal turns of the cochlea. The extent of strial pigmentation was unrelated to the EP value, which was measured from the basal turn only. Compound action potential (CAP) responses recorded from ears with an EP were variable and they showed greatly raised thresholds or were absent in all ears where the EP was close to zero. In controls, melanocytes in the vestibular part of the ear were found in the utricle, crus commune, and ampullae, whereas in many mutants only one or two of these regions were pigmented. There was a broad correlation between pigmentation of the stria and pigmentation of the vestibular region but this was not absolute. All W41/Wx, Wsh/Wsh, and W41/W41 mutants had some pigment on the pinna but, in contrast to controls where melanocytes were found in the epidermis and dermis of the pinna, pigment cells were reduced in number and generally restricted to the dermis. Injection of normal neural crest cells into 9.5-day-old mutant embryos increased the extent of skin pigmentation on the head and coat of adult chimeras and was associated with a small increase in the proportion of pigmented strias.

Crossing the SJL lambda locus into kappa-knockout mice reveals a dysfunction of the lambda 1-containing immunoglobulin receptor in B cell differentiation.

Mice of the SJL strain produce approximately 50 times less serum lambda 1 immunoglobulin light chains than other mouse strains. The defect is genetically linked to the lambda locus, but it is unknown whether it is due to regulatory alterations or known structural changes. We find no mutation in the SJL lambda 3-1 enhancer which regulates both lambda 1 and lambda 3. To investigate the defect further, the production of lambda light chains was amplified by crossing SJL with kappa-knockout mice. In kappa-knockout mice with the wildtype lambda locus (kappa -/- lambda +/+), the majority of serum light chains are lambda 1. In contrast, kappa-knockout mice with the SJL lambda locus (kappa -/- lambda s/s) show a pronounced expression of lambda 2 and lambda 3, with only some expression of lambda 1. The results show that the SJL defect is lambda 1 specific, since the linked lambda 3 expression is normal. As the transcription and rearrangement of lambda 1 appear normal in SJL, the defective lambda 1 synthesis is most likely due to a point mutation in the lambda 1 constant region resulting in a glycine to valine substitution. At the cellular level, in kappa-knockout mice with the SJL lambda locus there are fewer immature, and especially mature, lambda 1 B cells and the production of lambda 1 plasma cells is strongly inhibited. The lambda 1 specificity of the defect suggests that the point mutation in SJL C lambda 1 creates an immunoglobulin receptor complex which is dysfunctional in B cell differentiation.

Generation of pigmented stripes in albino mice by retroviral marking of neural crest melanoblasts.

The pigment cells of the skin are derived from melanoblasts which originate in the neural crest. The dorsoventral migration of melanoblasts has been visualized in pigment stripes seen in aggregation chimeras, and the width of these bands has suggested that the entire pigmentation of the coat is derived from a small number of founder cells. We have generated mosaic mice by marking single melanoblasts in utero to gain information on the clonal history of pigment-forming cells. A retroviral vector carrying the human tyrosinase gene was constructed and microinjected into neurulating albino mouse embryos. Albino mice are devoid of pigmentation due to deficiency of tyrosinase. Thus, transduction of the wild-type gene into the otherwise normal melanoblasts should rescue the mutant phenotype, giving rise to patches of pigmentation, which correspond to the area colonized by the mitotic progeny of a marked clone. Mosaic animals derived from the injected embryos indeed showed pigmented bands with a width strikingly similar to the 'standard' stripes seen in aggregation chimeras. These results are consistent with the notion that the unit width bands seen in aggregation chimeras represent the clonal progeny of a single melanoblast and verify Mintz's (1967) conclusion that a few founder melanoblasts give rise to coat pigmentation. The pigment cells of the eye are of dual origin: the melanocytes in choroid and outer layer of the iris are derived from the neural crest and those in the pigment layer of the retina from the neuroepithelium of the optic cup. Marked clones in both lineages were observed in the eyes of many mosaic animals.

Introduction of a selectable gene into primitive stem cells capable of long-term reconstitution of the hemopoietic system of W/Wv mice.

We have used the random chromosomal integration sites of retrovirus vectors as unique clonal markers to analyze cell lineage relationships within the hemopoietic stem cell hierarchy. Using a high efficiency protocol for retrovirus-mediated gene transfer, anemic W/Wv mutant mice were reconstituted with bone marrow cells infected with a NEO vector. Analysis of the DNA from bone marrow, thymus, and spleen of these reconstituted W/Wv mice indicated insertion of the vector into primitive pluripotent stem cells capable of producing both myeloid and lymphoid progeny as well as into more committed stem cells apparently restricted to either the myeloid or lymphoid lineages. The neo gene was also expressed in these mice, as they contained a variety of G418 resistant in vitro colony-forming cells. These results demonstrate high-efficiency gene transfer and expression in primitive hemopoietic stem cells and provide a direct approach for analyzing the hemopoietic stem cell hierarchy.

Phenotypic rescue of mutant brown melanocytes by a retrovirus carrying a wild-type tyrosinase-related protein gene.

A mouse cDNA for the developmentally controlled, melanocyte-specific protein, tyrosinase-related protein 1 (TRP-1), was previously cloned and reported to show genetic linkage with the coat-colour locus brown (b) on mouse chromosome 4. The cDNA has been inserted into a retroviral vector derived from Moloney murine leukaemia virus, under the control of the human histone H4 promoter. This vector was used to infect melanocytes of the immortal line melan-b, which are homozygous for the b mutation and which display light brown pigmentation in culture. Infected cultures containing between 0.2 and 2 copies of provirus per cell displayed an altered phenotype: 20-50% of cells now had the black to dark brown colour characteristic of cultured wild-type (Black, B/B) mouse melanocytes. Thus the TRP-1 gene complements the brown mutation. We conclude that TRP-1 is the product of the wild-type b-locus.