Mouse mutants from chemically mutagenized embryonic stem cells.

The drive to characterize functions of human genes on a global scale has stimulated interest in large-scale generation of mouse mutants. Conventional germ-cell mutagenesis with N-ethyl-N-nitrosourea (ENU) is compromised by an inability to monitor mutation efficiency, strain and interlocus variation in mutation induction, and extensive husbandry requirements. To overcome these obstacles and develop new methods for generating mouse mutants, we devised protocols to generate germline chimaeric mice from embryonic stem (ES) cells heavily mutagenized with ethylmethanesulphonate (EMS). Germline chimaeras were derived from cultures that underwent a mutation rate of up to 1 in 1,200 at the Hprt locus (encoding hypoxanthine guanine phosphoribosyl transferase). The spectrum of mutations induced by EMS and the frameshift mutagen ICR191 was consistent with that observed in other mammalian cells. Chimaeras derived from ES cells treated with EMS transmitted mutations affecting several processes, including limb development, hair growth, hearing and gametogenesis. This technology affords several advantages over traditional mutagenesis, including the ability to conduct shortened breeding schemes and to screen for mutant phenotypes directly in ES cells or their differentiated derivatives.

Germline genome protection: implications for gamete quality and germ cell tumorigenesis.

BACKGROUND: Germ cells have a unique and critical role as the conduit for hereditary information and therefore employ multiple strategies to protect genomic integrity and avoid mutations. Unlike somatic cells, which often respond to DNA damage by arresting the cell cycle and conducting DNA repair, germ cells as well as long-lived pluripotent stem cells typically avoid the use of error-prone repair mechanisms and favor apoptosis, reducing the risk of genetic alterations. Testicular germ cell tumors, the most common cancers of young men, arise from pre-natal germ cells. OBJECTIVES: To summarize the current understanding of DNA damage response mechanisms in pre-meiotic germ cells and to discuss how they impact both the origins of testicular germ cell tumors and their remarkable responsiveness to genotoxic chemotherapy. MATERIALS AND METHODS: We conducted a review of literature gathered from PubMed regarding the DNA damage response properties of testicular germ cell tumors and the germ cells from which they arise, as well as the influence of these mechanisms on therapeutic responses by testicular germ cell tumors. RESULTS AND DISCUSSION: This review provides a comprehensive evaluation of how the developmental origins of male germ cells and their inherent germ cell-like DNA damage response directly impact the development and therapeutic sensitivity of testicular germ cell tumors. CONCLUSIONS: The DNA damage response of germ cells directly impacts the development and therapeutic sensitivity of testicular germ cell tumors. Recent advances in the study of primordial germ cells, post-natal mitotically dividing germ cells, and pluripotent stem cells will allow for new investigations into the initiation, progression, and treatment of testicular germ cell tumors.

High-frequency germ line gene conversion in transgenic mice.

Gene conversion is the nonreciprocal transfer of genetic information between two related genes or DNA sequences. It can influence the evolution of gene families, having the capacity to generate both diversity and homogeneity. The potential evolutionary significance of this process is directly related to its frequency in the germ line. While measurement of meiotic inter- and intrachromosomal gene conversion frequency is routine in fungal systems, it has hitherto been impractical in mammals. We have designed a system for identifying and quantitating germ line gene conversion in mice by analyzing transgenic male gametes for a contrived recombination event. Spermatids which undergo the designed intrachromosomal gene conversion produce functional beta-galactosidase (encoded by the lacZ gene), which is visualized by histochemical staining. We observed a high incidence of lacZ-positive spermatids (approximately 2%), which were produced by a combination of meiotic and mitotic conversion events. These results demonstrate that gene conversion in mice is an active recombinational process leading to nonparental gametic haplotypes. This high frequency of intrachromosomal gene conversion seems incompatible with the evolutionary divergence of newly duplicated genes. Hence, a process may exist to uncouple gene pairs from frequent conversion-mediated homogenization.

Molecular cloning and genetic mapping of the t complex responder candidate gene family.

Male transmission ratio distortion (TRD) is a property of mouse t haplotypes requiring the t complex responder locus (Tcr). Tcr maps to the central region of t haplotypes, and is embedded within a series of large duplicated tracts of DNA known as "T66 elements." In previous work, a family of genes (the "T66" genes) was identified within this region that encodes male germ cell-specific transcripts. Genetic and molecular data indicate that one of these genes represents Tcr. Here, we describe the molecular cloning of the four members of the T66 gene family, the genetic mapping of these genes to three adjacent t haplotype loci, and comparative restriction enzyme analysis of the genes. The results indicate that these genes are highly similar to one another, and were created by recent, complex duplication events. This suggests that a minor alteration(s) could have been responsible for conferring "mutant" responder activity upon Tcr, while the other homologs retained "wild-type" biochemical function. In addition, we have identified and mapped three T66 genes in wild-type t complexes. They reside in two separate loci at the opposite ends of the proximal t complex inversion, and are separated by at least 3 cM.

Gene conversion between unlinked sequences in the germline of mice.

Gene conversion between homologous sequences on non-homologous chromosomes (ectopic gene conversion) is remarkably frequent in fungi. It is thought to be a consequence of genome-wide homology scanning required to form synapses between homologous chromosomes. This activity provides a mechanism for concerted evolution of dispersed genes. Technical obstacles associated with mammalian systems have hitherto precluded investigations into ectopic gene conversion in the mammals. Here, we describe a binary transgenic mouse system to detect ectopic gene conversion in mice. Conversion events are visualized by histochemical staining of spermatids, and corroborated by polymerase chain reaction amplification of transgenes in spermatozoa. The results show that conversion between unliked, hemizygous lacZ transgenes is frequent in the male germline, ranging from 0.1 to 0.7% of spermatids. Genomic location may affect the susceptibility to recombination, since the frequency varied between lines. The results suggest that homologous genes can undergo concerted evolution despite being genomically dispersed. However, mechanisms may exist to modulate this activity, enabling the divergence of duplicated genes.

Physical mapping of male fertility and meiotic drive quantitative trait loci in the mouse t complex using chromosome deficiencies.

The t complex spans 20 cM of the proximal region of mouse chromosome 17. A variant form, the t haplotype (t), exists at significant frequencies in wild mouse populations and is characterized by the presence of inversions that suppress recombination with wild-type (+) chromosomes. Transmission ratio distortion and sterility are associated with t and affect males only. It is hypothesized that these phenomena are caused by trans-acting distorter/sterility factors that interact with a responder locus (Tcr(t)) and that the distorter and sterility factors are the same because homozygosity of the distorters causes male sterility. One factor, Tcd1, was previously shown to be amorphic using a chromosome deletion. To overcome limitations imposed by recombination suppression, we used a series of deletions within the t complex in trans to t chromosomes to characterize the Tcd1 region. We find that the distorter activity of Tcd1 is distinct from a linked sterility factor, originally called tcs1. YACs mapped with respect to deletion breakpoints localize tcs1 to a 1.1-Mb interval flanked by D17Aus9 and Tctex1. We present evidence for the existence of multiple proximal t complex regions that exhibit distorter activity. These studies demonstrate the utility of chromosome deletions for complex trait analysis.

Narrowing the critical regions for mouse t complex transmission ratio distortion factors by use of deletions.

Previously a deletion in mouse chromosome 17, T(22H), was shown to behave like a t allele of the t complex distorter gene Tcd1, and this was attributed to deletion of this locus. Seven further deletions are studied here, with the aim of narrowing the critical region in which Tcd1 must lie. One deletion, T(30H), together with three others, T(31H), T(33H), and T(36H), which extended more proximally, caused male sterility when heterozygous with a complete t haplotype and also enhanced transmission ratio of the partial t haplotype t(6), and this was attributed to deletion of the Tcd1 locus. The deletions T(29H), T(32H), and T(34H) that extended less proximally than T(30H) permitted male fertility when opposite a complete t haplotype. These results enabled narrowing of the critical interval for Tcd1 to between the markers D17Mit164 and D17Leh48. In addition, T(29H) and T(32H) enhanced the transmission ratio of t(6), but significantly less so than T(30H). T(34H) had no effect on transmission ratio. These results could be explained by a new distorter located between the breakpoints of T(29H) and T(34H) (between T and D17Leh66E). It is suggested that the original distorter Tcd1 in fact consists of two loci: Tcd1a, lying between D17Mit164 and D17Leh48, and Tcd1b, lying between T and D17Leh66E.

Deletion mapping of the head tilt (het) gene in mice: a vestibular mutation causing specific absence of otoliths.

Head tilt (het) is a recessive mutation in mice causing vestibular dysfunction. Homozygotes display abnormal responses to position change and linear acceleration and cannot swim. However, they are not deaf. het was mapped to the proximal region of mouse chromosome 17, near the T locus. Here we report anatomical characterization of het mutants and high resolution mapping using a set of chromosome deletions. The defect in het mutants is limited to the utricle and saccule of the inner ear, which completely lack otoliths. The unique specificity of the het mutation provides an opportunity to better understand the development of the vestibular system. Complementation analyses with a collection of embryonic stem (ES)- and germ cell-induced deletions localized het to an interval near the centromere of chromosome 17 that was indivisible by recombination mapping. This approach demonstrates the utility of chromosome deletions as reagents for mapping and characterizing mutations, particularly in situations where recombinational mapping is inadequate.

Reciprocal mouse and human limb phenotypes caused by gain- and loss-of-function mutations affecting Lmbr1.

The major locus for dominant preaxial polydactyly in humans has been mapped to 7q36. In mice the dominant Hemimelic extra toes (Hx) and Hammertoe (Hm) mutations map to a homologous chromosomal region and cause similar limb defects. The Lmbr1 gene is entirely within the small critical intervals recently defined for both the mouse and human mutations and is misexpressed at the exact time that the mouse Hx phenotype becomes apparent during limb development. This result suggests that Lmbr1 may underlie preaxial polydactyly in both mice and humans. We have used deletion chromosomes to demonstrate that the dominant mouse and human limb defects arise from gain-of-function mutations and not from haploinsufficiency. Furthermore, we created a loss-of-function mutation in the mouse Lmbr1 gene that causes digit number reduction (oligodactyly) on its own and in trans to a deletion chromosome. The loss of digits that we observed in mice with reduced Lmbr1 activity is in contrast to the gain of digits observed in Hx mice and human polydactyly patients. Our results suggest that the Lmbr1 gene is required for limb formation and that reciprocal changes in levels of Lmbr1 activity can lead to either increases or decreases in the number of digits in the vertebrate limb.

Molecular analysis of gene conversion in spermatids from transgenic mice.

Investigations into the mechanisms and properties of gene conversion in mammals are greatly restricted by the inability to recover all the products of a meiosis. Additionally, the study of this process has been hampered by the lack of visible markers to detect gene conversion, especially when the events are rare. In previous work, we developed a transgenic system for detection and quantitation of gene conversion events in the germline of mice (Murti, J.R., Bumbulis, M., Schimenti, J.C., 1992. High frequency germline gene conversion in transgenic mice. Mol. Cell. Biol. 12, 2545-2552) that could be exploited as an assay for recombinogenic chemicals (Murti, J.R, Schimenti, K.J., Schimenti, J.C., 1994. A recombination-based transgenic mouse system for genotoxicity testing. Mutat. Res. 307, 583-595). A specific intrachromosomal gene conversion event between two complementarily defective lacZ genes resulted in the production of beta-galactosidase in spermatids, enabling a measurement of conversion frequency. Here, we report that the anticancer drug, cisplatin, increased gene conversion in meiotic stage cells in these transgenic mice. Furthermore, a method was developed for direct molecular analysis of transgene conversion events in single or pooled lacZ-positive spermatids. The ability to identify gametes that have undergone a rare gene conversion event, followed by molecular amplification of the recombinant gene, should make it possible to investigate the mechanisms of genetic recombination in mammals in greater detail than previously possible.

Homologous recombinational repair proteins in mouse meiosis.

Eukaryotic meiotic recombination requires numerous biochemical processes, including break initiation, end resection, strand invasion and heteroduplex formation, and, finally, crossover resolution. In this review, we discuss primarily those proteins involved in the initial stages of homologous recombination, including SPO11, MRE11, RAD50, NBS1, DMC1, RAD51, RAD51 paralogs, RAD52, RPA, RAD54, and RAD54B. Focusing on the mouse as a model organism, we discuss what is known about the conserved roles of these proteins in vertebrate somatic cells and in mammalian meiosis. We consider such information as gene expression in gonadal tissue, protein localization patterns on chromosomal cores in meiocyte nuclei, and information gleaned from mouse models.

New mouse genetic models for human contraceptive development.

Genetic strategies for the post-genomic sequence age will be designed to provide information about gene function in a myriad of physiological processes. Here an ENU mutagenesis program (http://reprogenomics.jax.org) is described that is generating a large resource of mutant mouse models of infertility; male and female mutants with defects in a wide range of reproductive processes are being recovered. Identification of the genes responsible for these defects, and the pathways in which these genes function, will advance the fields of reproduction research and medicine. Importantly, this program has potential to reveal novel human contraceptive targets.

Function of untranslated regions in the mouse spermatogenesis-specific gene Tcp10 evaluated in transgenic mice.

The mouse Tcp10 genes are transcribed exclusively in male germ cells and display multiple 5' and 3' untranslated variations generated by alternative splicing and polyadenylation signal usage. To investigate the possible role of untranslated sequences in the regulation of these genes, chimeric expression constructs with or without endogenous 5' and 3' untranslated sequences were generated and used to make transgenic mice. Analysis of these animals showed that the untranslated sequences have no effect on the transcription or translation of an attached lacZ reporter gene, thereby implying these sequences are dispensible. However, the endogenous pattern of polyadenylation site usage was altered when Tcp10 3' untranslated sequences were linked to lacZ, indicating that internal coding sequence can influence recognition of polyadenylation signals in testis. The characteristics of alternative splicing and polyadenylation signal variability reflects a common theme of promiscuity in testicular gene expression.

Vestibular responses to linear acceleration are absent in otoconia-deficient C57BL/6JEi-het mice.

UNLABELLED: Vestibular evoked potentials (VsEPs) were measured in normal mice and in mice homozygous for the head tilt mutation (het/het, abbr. het). The het mice lack otoconia, the inertial mass critical for natural stimulation of inner ear gravity receptors. Our findings demonstrate that vestibular neural responses to pulsed linear acceleration are absent in het mice. THE RESULTS: (1) confirm that adequate sensory stimuli fail to activate gravity receptors in the het model; and (2) serve as definitive evidence that far-field vestibular responses to pulsed linear acceleration depend critically on otolith end organs. The C57BL/6JEi-het mouse may be an excellent model of gravity receptor sensory deprivation.

A recombination-based transgenic mouse system for genotoxicity testing.

It is well established that mutagens induce recombination in cultured cells and experimental organisms. Presumably, this is a consequence of the DNA-damage-triggering cellular-repair mechanisms. The relationship between recombination and mutagenicity has been exploited in submammalian organisms, such as yeast, to assay the ability of chemical agents and radiation to induce a form of recombination called gene conversion--the non-reciprocal transfer of genetic information. This work has demonstrated the efficacy of predicting mutagenicity on the basis of recombination induction. Here, we describe the utilization of a transgenic mouse system for efficient detection of germ-line gene-conversion events as a mutagen-screening tool. These mice contain two mutually defective reporter (lacZ) genes under the regulatory control of a spermatogenesis-specific promoter. A particular intrachromosomal gene conversion event must occur for the generation of functional lacZ activity. Conversion events are visualized by histochemical staining or flow cytometric analysis of transgenic spermatids. The highly mutagenic compound chlorambucil induced a several fold percentage-wise increase of lacZ-positive spermatids, whereas acrylamide, a weak genotoxin, produced no marked increase in converted spermatids. The results indicate that recombination-based transgenic mouse models for genotoxin screening present a viable option for inexpensive and rapid whole-animal mutagen testing. The particular mice we describe may ultimately prove to be a useful tool for identifying agents which can cause heritable genetic mutations in humans.

Role of DNA damage response pathways in preventing carcinogenesis caused by intrinsic replication stress.

Defective DNA replication can result in genomic instability, cancer and developmental defects. To understand the roles of DNA damage response (DDR) genes on carcinogenesis in mutants defective for core DNA replication components, we utilized the Mcm4(Chaos3/Chaos3) ('Chaos3') mouse model that, by virtue of an amino-acid alteration in MCM4 that destabilizes the MCM2-7 DNA replicative helicase, has fewer dormant replication origins and an increased number of stalled replication forks. This leads to genomic instability and cancer in most Chaos3 mice. We found that animals doubly mutant for Chaos3 and components of the ataxia telangiectasia-mutated (ATM) double-strand break response pathway (Atm, p21/Cdkn1a and Chk2/Chek2) had decreased tumor latency and/or increased tumor susceptibility. Tumor latency and susceptibility differed between genetic backgrounds and genders, with females demonstrating an overall greater cancer susceptibility to Atm and p21 deficiency than males. Atm deficiency was semilethal in the Chaos3 background and impaired embryonic fibroblast proliferation, suggesting that ATM drug inhibitors might be useful against tumors with DNA replication defects. Hypomorphism for the 9-1-1 component Hus1 did not affect tumor latency or susceptibility in Chaos3 animals, and tumors in these mice did not exhibit impaired ATR pathway signaling. These and other data indicate that under conditions of systemic replication stress, the ATM pathway is particularly important both for cancer suppression and viability during development.

Mutagenesis-generated mouse models of human infertility with abnormal sperm.

BACKGROUND: The aetiology of human male fertility, with impairment of sperm number, motility and morphology (oligoasthenoteratozoospermia), has been difficult to understand, partly for lack of animal models. METHODS: An ethylnitrosourea (ENU) mutagenesis strategy has been successful in producing heritable gene mutations with phenotypes similar to human male infertility, and here, we describe three independent ENU-induced mutations that cause a phenotype of oligoasthenoteratozoospermia in mice. RESULTS: The loci identified by these three mutations are designated swm2, repro2 and repro3. All mutant males were characterized by low sperm concentration, poor sperm morphology and negligible motility, but the infertile males were apparently normal in other respects. Sperm from mutant males failed to fertilize oocytes in vitro. Ultrastructural analyses revealed varied abnormalities apparent in both testicular spermatids and epididymal sperm. Genetic mapping placed the swm2 gene on chromosome 7, the repro2 gene on chromosome 5 and the repro3 gene on chromosome 10. CONCLUSION: The single-gene mutations caused complex and non-specific sperm pathologies, a point with important implications for managing cases of human male infertility. The ultimate identification of the loci for the mutations causing these phenotypes will clarify aetiology of complex syndromes of infertility with sperm abnormalities consistent with oligoasthenoteratozoospermia.

ORFless, intronless, and mutant transcription units in the mouse t complex responder (Tcr) locus.

Mouse t haplotypes exhibit transmission ratio distortion (TRD), whereby heterozygous males (+/t) transmit the t chromosome to nearly all offspring. TRD is mediated by the t complex responder locus (Tcr), whose transmission is elevated above Mendelian levels by additive contribution of several t haplotype-encoded quantitative trait loci (QTLs) called t complex distorters (Tcd1-Tcd5). The entire genetically defined Tcr interval has been cloned and consists of under 200 kb. This interval is one of three large duplication units in t haplotypes that retain high levels of similarity. The cis-active nature of Tcr raises the possibility that it is not a protein-encoding gene, but another anomaly such as a structural anomaly of chromatin. To further investigate the Tcr-critical interval, a 30-kb region upstream of the Tcp10b(t) gene (a testis-expressed former candidate for Tcr) was sequenced, along with the duplicated paralogous region associated with Tcp10c(t), which lies immediately adjacent but outside the Tcr interval. Several genes or transcriptional units were identified, including the 3' end of ribosomal s6 kinase (Rsk3); two apparently intronless and ORF-less genes; and Gpr31, an intronless, putative G-protein coupled receptor. While the 30-kb regions were 98% identical, the Gpr31 paralog from the Tcr-critical region (Gpr31b(t)) contained an in-frame 210-bp deletion that disrupted two of the seven predicted transmembrane domains. Furthermore, an intronless and ORFless gene from this interval, Trex1b(t), contained a 4-bp deletion that distinguished it from all other homologs in t haplotypes and wild-type chromosomes. Although it is unknown whether any of these genes are involved in TRD, their discovery raises new possibilities regarding the nature of Tcr, including a model whereby it might function as an RNA rather than a protein or chromatin anomaly.