A systematic, genome-wide, phenotype-driven mutagenesis programme for gene function studies in the mouse.

As the human genome project approaches completion, the challenge for mammalian geneticists is to develop approaches for the systematic determination of mammalian gene function. Mouse mutagenesis will be a key element of studies of gene function. Phenotype-driven approaches using the chemical mutagen ethylnitrosourea (ENU) represent a potentially efficient route for the generation of large numbers of mutant mice that can be screened for novel phenotypes. The advantage of this approach is that, in assessing gene function, no a priori assumptions are made about the genes involved in any pathway. Phenotype-driven mutagenesis is thus an effective method for the identification of novel genes and pathways. We have undertaken a genome-wide, phenotype-driven screen for dominant mutations in the mouse. We generated and screened over 26,000 mice, and recovered some 500 new mouse mutants. Our work, along with the programme reported in the accompanying paper, has led to a substantial increase in the mouse mutant resource and represents a first step towards systematic studies of gene function in mammalian genetics.

Identification of a mutation in the MP19 gene, Lim2, in the cataractous mouse mutant To3.

PURPOSE: Lim2, the gene encoding the second most abundant lens specific integral membrane protein, MP19, has recently been proposed as an ideal candidate gene for the cataractous mouse mutant, To3. The aim of this study was to screen the Lim2 gene in the To3 mutant for a genetic lesion that was correlated and consistent with the mutant phenotype. METHODS: Genomic DNA was isolated from both normal mouse parental strains as well as the heterozygous and homozygous To3 cataract mutant. PCR was used to generate overlapping fragments of the entire Lim2 gene from these DNAs. The coding regions, including splice junctions and the translational termination site, of these fragments were then sequenced. RESULTS: A single G -> T transversion was identified within the first coding exon of the Lim2 gene in the To3 mutant DNA. This DNA change results in the nonconservative substitution of a valine for the normally encoded glycine at amino acid 15 of the MP19 polypeptide. CONCLUSIONS: The identified genetic lesion in the Lim2 gene of the cataractous mouse mutant, To3, confirms Lim2 as an ideal candidate gene. Future transgenic experiments should provide proof or disproof of a causative relationship between the identified mutation and the cataractous phenotype. These studies indicate that MP19 may play an important role in both normal lens development and cataractogenesis, and warrants more intense investigation of its role within the ocular lens.

A search for strain differences in response of mice to mutagenesis by thio-TEPA.

After treatment of mice with thio-TEPA Malashenko and colleagues found differences among inbred strains in yield of dominant lethals and of chromosome aberrations in bone marrow, which they attributed to genes affecting repair. An attempt was made to confirm this work by comparing yields of dominant lethals in different strains of females mated to the same strain of males. However, no differences were found, all strain combinations giving 42-49% dominant lethals after a dose of 2 mg/kg thio-TEPA to late spermatids. Thus, the existence of genetic differences in repair of thio-TEPA induced lesions between strains CBA and C57BL/6J and between C3H/He and 101/H is not confirmed. Possible reasons for the discrepant results are discussed.

Long-term storage of eight-cell mouse embryos at - 196 degrees C.

Stocks of mutant mice have been reestablished from eight-cell embryos stored in liquid nitrogen for varying periods up to 11 years, and no evidence has been found of deterioration of survival with time of storage. Also, studies on the simulated cumulative effect of background radiation during storage failed to find any detrimental effect when embryos were exposed to the equivalent of about 2000 years of background radiation. However, in some cases embryos that carry mutant genes or chromosome anomalies tend to survive the freezing and thawing procedure less well than F1 hybrid embryos. Although this effect is probably independent of storage time, recent improvements in technique upon embryonic survival are to be welcomed.

Cryoconservation--archiving for the future.

Mouse genetics is set to play a pivotal role in the key post-genome challenge-the study of mammalian gene function. Addressing this challenge will involve the development and application of systematic mutagenesis approaches. The expanding mouse mutant resource that will result threatens to overwhelm the currently available animal facility space. Cryopreservation of both mouse embryos and spermatozoa is currently widely employed for the efficient archiving of mouse stocks. Distribution and dissemination of new and existing mouse strains is simplified by the availability of extensive frozen archives. Also, the availability of archives of frozen spermatozoa provides a potential powerful route for the production of backcross progeny for rapid genetic mapping. Moreover, frozen oocytes and ovaries may offer a valuable addition to the current cryopreservation approaches. Comprehensive mouse mutant archives will provide an essential resource for mammalian genetics throughout the 21(st) century.

Reduced reproductive performance in androgen-resistant Tfm/Tfm female mice.

Androgen-resistant female mice (Tfm/Tfm) homozygous for the mutant gene Tfm were bred by making use of males chimaeric for the Tfm gene. All seven Tfm/Tfm females found were fertile, confirming that a normal level of androgen receptor protein is not essential for reproduction in female mice. However, when five of the seven were studied throughout their reproductive life they proved to have impaired reproductive performance and premature cessation of reproduction. No impairment of reproduction was seen in heterozygous Tfm/+ females. The ovarian histology suggested that in Tfm/Tfm the normal ageing processes were accelerated. This work is consistent with the work of others in that androgen is involved in the control of follicular maturation and atresia, and that the effect is mediated by the androgen receptor coded by the Tfm locus.

Further studies on the effect of radiation during the storage of frozen 8-cell mouse embryos at -196 degrees C.

Frozen 8-cell mouse embryos were treated with radiation doses of 0, 10, 50, 100 or 200 cGy gamma-rays at a dose rate of congruent to 5 cGy/day. After thawing the embryos were scored for normal morphological appearance and for development to morulae and blastocysts after 24 h in culture. Embryos from each treatment were then separately transferred to the uteri of pseudopregnant foster mothers which were killed at Day 14 of pregnancy. There was no effect of radiation on morphological appearance, development to morulae and blastocysts, implantation rate, or on the ratio of live fetuses to the number of transferred embryos. As there appeared to be no detrimental effect of up to 200 cGy on frozen 8-cell mouse embryos and, as this is the equivalent of congruent to 2000 years of background radiation, it is concluded that normal levels of background radiation would not be a hazard to the long-term storage of mammalian embryos.

Genome cryopreservation: a valuable contribution to mammalian genetic research.

Mouse embryo banking has become an important asset to geneticists. Individual laboratories can now maintain a far greater diversity of stocks than by conventional breeding alone. Also, many mutations that in the past would have been discarded due to lack of space, can now be preserved for future use. Recent advances in cryopreservation techniques have simplified procedures and, in certain cases, resulted in increased rates of survival.

Large numbers of mice established by in vitro fertilization with cryopreserved spermatozoa: implications and applications for genetic resource banks, mutagenesis screens, and mouse backcrosses.

Recent advances in the methodologies for cryopreservation of mouse spermatozoa have opened up a number of opportunities for mouse geneticists. We have investigated various applications for this relatively new technology and have explored the potential of sperm freezing coupled with IVF for archiving, stock building, and the rapid establishment of backcrosses. Firstly, we investigated the use of sperm freezing for the archiving of (C3H/HeH x BALB/c)F(1) progeny from a large-scale mutagenesis program. We have demonstrated that it is possible to establish efficient, comprehensive, and deep archives and that potentially thousands of offspring can be derived from the frozen spermatozoa of a single mutant male mouse. Secondly, we examined the efficacy of sperm freezing for a number of other genotypes. For at least some genotypes, frozen spermatozoa can be utilized to rapidly build stock far more quickly than by conventional methods. Finally, we demonstrated that it is feasible to use frozen spermatozoa from the mouse mutant archive for the rapid generation of mutant backcross progeny.

Incidence of chromosome anomalies in first-cleavage mouse embryos obtained from frozen-thawed oocytes fertilized in vitro.

To assess the effect of low temperature storage on mouse oocytes we (1) examined the capacity for normal development of embryos derived from frozen oocytes fertilized in vitro after transfer to pseudopregnant foster mothers and (2) analyzed the chromosome complement at the first cleavage division. Fewer frozen than control oocytes were fertilized (36% vs 66%), but after embryo transfer the proportion of fertilized eggs that implanted (67-68%) and formed normal foetuses (50-53%) was similar in the two groups. Freezing did not affect the observed incidence of aneuploidy (1.5-3.3%). The frequency of polyploid embryos derived from frozen oocytes was almost double that of controls (15.8% vs 8.5%), but it is unclear whether this is a real effect of freezing or is an artifact produced by the chromosome preparation technique.

Age related reactivation of an X-linked gene.

We have investigated age-related reactivation of the X chromosome by devising a model in which reactivation of a single gene in one cell among many can be identified. We have used mice with an X-autosomal translocation giving consistent non-random inactivation of the normal X (as judged by biochemical and cytogenetic techniques), that also carry a defective form of a histochemically demonstrable X-linked enzyme. When the gene for the normal enzyme was located on the inactivated normal X a uniformly negative histochemical picture would be predicted in doubly heterozygous animals. A very small proportion of enzyme-positive cells was found in young animals. This proportion increased very significantly with age, but the patch size did not change, showing that the result was not due to preferential division of enzyme-positive cells, but was instead due to the conversion of previously enzyme-negative to enzyme-positive cells. These observations provide the first evidence with a true X-linked gene for an age-related decrease in the stability of the X-inactivation mechanism.

Doublefoot: a new mouse mutant affecting development of limbs and head.

The mutant doublefoot, Dbf, of the mouse arose spontaneously, and was shown to be inherited as an autosomal dominant, mapping 9-13 cM proximal to leaden, In, on chromosome 1 and showing no recombination with the microsatellite markers D1Mit24 and D1Mit77. In heterozygotes the phenotype includes many extra toes on all four feet, and the tibia and fibula may be reduced and bowed. The head is shortened and broad and the eyes are held half-closed, and some animals develop hydrocephalus. The tail is kinked and abnormally thick, and the soles of the feet are swollen. Growth is retarded, viability is reduced, and reproduction is impaired in both sexes. Only about 30% of males are normally fertile, and testis weights and sperm counts may be reduced, although this appears not to be the main cause of poor fertility. In females vaginal opening is delayed and oestrous cycles are irregular, although the animals appear to respond to gonadotrophic hormones. Crosses of Dbf/+ x Dbf/+ are very poorly fertile. Prenatally, Dbf/+ heterozygotes can first be recognized at 11 1/2 days gestation by abnormally broad fore limb buds. Putative Dbf/Dbf homozygotes at 12 1/2 days have similar limbs defects and also split face, due to failure of the maxillae to fuse in the midline. Some homozygotes and a few putative heterozygotes have cranioschisis. At 13 1/2 days, the heads of homozygotes tend to bulge in the frontal region and a bleb of clear fluid is visible medially. At 14 1/2 days Dbf/Dbf fetuses may have oedema and some are dead. From 15 1/2 days onwards no live Dbf/Dbf fetuses have been found. The gene maps close to the locus of Pax3, but crossovers between Dbf and Pax3 have been found, ruling out the possibility that a gain-of-function mutation in Pax3 might be involved.

The scurfy mouse mutant has previously unrecognized hematological abnormalities and resembles Wiskott-Aldrich syndrome.

The X chromosome-linked scurfy (sf) mutant of the mouse is recognized by the scaliness of the skin from which the name is derived and results in death of affected males at about 3-4 weeks of age. Consideration of known man-mouse homologies of the X chromosome prompted hematological studies, which have shown that the blood is highly abnormal. The platelet and erythrocyte counts are both reduced and become progressively lower relative to normal as the disease progresses. There is gastrointestinal bleeding, and most animals appear to die of severe anemia. By contrast, the leukocyte count is consistently raised. Some animals showed signs of infection but it is not yet clear whether there is immunodeficiency. Other features include the scaly skin and apparently reduced lateral growth of the skin, conjunctivitis, and diarrhea in some animals. The mutant resembles Wiskott-Aldrich syndrome in man, which is characterized by thrombocytopenia, eczema, diarrhea, and immunodeficiency. The loci of the human and mouse genes lie in homologous segments of the X chromosome, although apparently in somewhat different positions relative to other gene loci. Scurfy differs from Wiskott-Aldrich syndrome in that scurfy males are consistently hypogonadal.

Ubiquitous expression and imprinting of Snrpn in the mouse.

Snrpn is known to be abundantly expressed in rodent brain and heart, and in two separate studies with neonatal mouse brain it has been shown to be maternally imprinted, that is, the maternal allele is normally repressed. We now provide evidence on the expression profile and imprinting status of Snrpn throughout development. Using RT-PCR, we have established that Snrpn is further expressed at low levels in lung, liver, spleen, kidney, skeletal muscle, and gonads. Moreover, using mice with only maternal copies of Snrpn (maternal duplication for the chromosome region involved and parthenogenotes), we have shown that the gene is imprinted in all of these tissues and, generally, from the time the gene is first expressed at 7.5 days gestation. In contrast to the findings made with the imprinted genes, Igf2, Ins1, and Ins2, there is no evidence of tissue-specific imprinting in the embryo with Snrpn. Nor, as found with Igf2 and Igf2r, is there evidence of a window of biallelic expression between the germ line imprint and the time of gene repression. The absence of Snrpn expression in early embryos contrasts with the findings in ES cells.

Two new cataract loci, Ccw and To3, and further mapping of the Npp and Opj cataracts in the mouse.

Many types of inherited early onset cataract are known in both human and mouse. Here we describe the mapping of two novel dominant cataract loci in the mouse genome. Cataract and curly whiskers, Ccw, maps to Chromosome 4, 3.1 +/- 1.1 cM distal to the b (brown) locus. Total opacity 3, To3, maps to Chromosome 7, 7.1 +/- 1.8 cM proximal to p (pink-eyed dilution). The map positions of two other dominant cataract mutants have now been refined by three-point crosses. Nuclear and posterior polar cataract, Npp, maps to the central part of Chromosome 5, 1.4 +/- 0.5 cM distal to We (dominant spotting-extreme, an allele at the Kit locus), and Opaque secondary fiber cell junctions, Opj, maps to the proximal region of Chromosome 16, 9.1 +/- 1.5 cM distal to the marker md (mahoganoid). While there are no obvious candidate genes in the vicinity of the Ccw, Npp, and Opj mutations, To3 lies remarkably close to the recently mapped Lim2 locus, which encodes lens intrinsic membrane protein 2, also called MP19.

Mapping of six dominant cataract genes in the mouse.

The mapping of six mouse autosomal dominant cataract mutations that were induced by mutagenic treatment with radiation or ethylnitrosourea is described. Three, with differing phenotypes, mapped on Chromosome 1 between the loci of fuzzy (fz) and leaden (ln) and close to the locus of the gamma-crystallin gene cluster. One of these, Cat-2t, had previously been shown to be a member of a group of five allelic mutants. In addition, the previously known mutant eye lens obsolescence, Elo, maps to the same point. There are thus now eight mutants that map to this point and that may involve mutations in one of the gamma-crystallin genes. In addition, one of these mutants may be a homologue of Coppock cataract in man, which also maps close to the gamma-crystallin locus. Of the three remaining mutants, one, with the suggested symbol Cat-5, mapped to the proximal region of Chromosome 10, 23.4 +/- 4.0 cM from downless (dl), a region with homology to human 6q. A second mutant, provisionally designated Opj, mapped on Chromosome 16, 8.2 +/- 3.9 cM from the marker mahoganoid (md). Thus, it possibly has a homologue on human 22q, a region in which one of the beta-crystallin loci is sited. A third mutant, provisionally designated Npp, mapped to Chromosome 5, 1.3 +/- 0.9 cM from the locus of W, and thus probably has a homologue on human Chromosome 4.