Peptidylarginine deiminase (PAD) 6 is essential for oocyte cytoskeletal sheet formation and female fertility.

Peptidylarginine deiminase 6 (PAD6) is an enzyme that is uniquely expressed in male and female germ cells. To study the function of this enzyme in vivo we generated mice deficient for PAD6. Here we show that inactivation of the PAD6 gene in mice leads to female infertility whereas male fertility is not affected. The absence of the PAD6 protein and consequently absence of citrullination activity in oocytes results in dispersal of the cytoskeletal sheets in oocytes, indicating an essential role of these germ cell-specific structures in zygote/embryo development. PAD6 deficient mice do not show any other overt phenotype. Thus, we identify citrullination as a new regulator of fertility.

Distinct spectra of somatic mutations accumulated with age in mouse heart and small intestine.

Somatic mutation accumulation has been implicated as a major cause of cancer and aging. By using a transgenic mouse model with a chromosomally integrated lacZ reporter gene, mutational spectra were characterized at young and old age in two organs greatly differing in proliferative activity, i.e., the heart and small intestine. At young age the spectra were nearly identical, mainly consisting of G. C to A.T transitions and 1-bp deletions. At old age, however, distinct patterns of mutations had developed. In small intestine, only point mutations were found to accumulate, including G.C to T.A, G.C to C.G, and A.T to C.G transversions and G.C to A.T transitions. In contrast, in heart about half of the accumulated mutations appeared to be large genome rearrangements, involving up to 34 centimorgans of chromosomal DNA. Virtually all other mutations accumulating in the heart appeared to be G.C to A.T transitions at CpG sites. These results suggest that distinct mechanisms lead to organ-specific genome deterioration and dysfunction at old age.

Quantitative correlation between radiation-induced mutagenesis in endogenous genes and transgenes of mouse spermatogonial stem cells.

In order to evaluate the pUR288-plasmid transgenic mouse model, utilizing the bacterial lacZ gene as the mutational target, radiation-induced mutagenesis was primarily analyzed in spermatogonial stem cells. A combined hydroxyurea (HU)-X-ray treatment protocol was used, known to sensitize dramatically the induction of mutations in endogenous genes. In the testes of untreated animals, a mutant frequency of 6.7 +/- 4.4 x 10(-5) was found. In animals treated with HU or X ray alone, moderate elevations were seen (factors of about 4 and 2 over untreated animal values). In testes of mice having received the HU + X-ray combination treatment, a mutant frequency of 63.0 +/- 36.1 x 10(-5) was found. The results obtained showed a good quantitative correlation between endogenous genes and the transgene, indicating the suitability of pUR288 transgenic mice for also efficiently recording radiation-induced genetic damage. Radiosensitization, seen in spermatogonial stem cells, was not observed in other studied organs such as spleen, brain, or lung.

Temporal and molecular characteristics of lacZ mutations in somatic tissues of transgenic mice.

In order to help establish criteria for optimizing protocols for in vivo mutation studies, lacZ transgenic mice (Muta mouse) were treated with five consecutive daily doses of ethylnitrosourea (50 mg/kg), sampled at times up to 55 days after treatment, and mutant frequencies and DNA sequences determined for liver and bone marrow. In the bone marrow, the mutant frequency rose very rapidly in the first 5 days after treatment to 34 times the control frequency. Subsequently, there was a brood peak where the mutant frequency did not vary significantly, although it did appear to begin to decline after 45 days. In contrast, in the liver, the peak mutant frequency (11 times the control frequency) was not achieved until 35 days, after which there appeared to be a slow decline up to 55 days, which was not statistically significant. Once the maximum mutant frequency was reached, the mutation spectra in the two tissues were indistinguishable. In contrast to the G:C-->A:T transitions in 5'-CpG sites characteristic of untreated mice, A:T-->T:A transversions and A:T-->G:C transitions were prominent in both liver and bone marrow of ENU-treated mice, suggesting the involvement of unrepaired O2- and O4-ethylthymine adducts. In addition, G:C-->T:A transversions were induced in liver. This study demonstrates the possibility that although tissues may have different mutation fixation times, a single mutation fixation time equal to the longest time may be appropriate for in vivo mutation studies, provided that the mutation frequency does not decline appreciably after the peak is reached. This study also illustrates the necessity of ensuring that mutation characteristics are determined after optimal fixation has occurred.

Evaluation of a plasmid-based transgenic mouse model for detecting in vivo mutations.

To study in vivo somatic mutations a C57BL/6 transgenic mouse model was constructed harboring multiple chromosomally integrated copies of the plasmid pUR288, which carried the lacZ reporter gene as the mutational target. We previously demonstrated that lacZ-containing plasmids could be rescued from their integrated state efficient enough to detect mutations in lacZ by positive selection. The smaller size of the plasmid vector, as compared with our earlier transgenic mouse model based on bacteriophage lambda vectors, should offer considerable advantages in terms of rescue efficiency and sensitivity to large size alterations in the lacZ gene. To evaluate the plasmid-based mouse model for its suitability to detect in vivo mutations, we determined mutant frequencies in different organs of untreated and ethyl nitrosourea (ENU)-treated animals using a new, improved protocol. The rescue efficiencies obtained were as high as 200,000/micrograms genomic DNA; millions of transformants could be obtained in one single experiment. The average spontaneous mutant frequency in four different organs of 4- to 8-week-old mice ranged from 4.41 to 6.82 x 10(-5), compared with a mutant frequency of the same plasmid grown in Escherichia coli of approximately 1 x 10(-5) or less. Single treatments with 100 and 250 mg ENU/kg body wt resulted in a 7- and 14-fold increase, respectively, in spleen mutant frequency at 14 days after i.p. administration of the alkylating agent. Restriction enzyme analysis showed that a considerable portion of spontaneous mutants were size changes varying from approximately 100 to 3000 bp. Some mutant plasmids contained mouse genomic sequences, which is indicative of large genetic rearrangement events involving the 3' flanking regions of the transgene cluster. Among the ENU-induced mutants, size changes comprised only a minor fraction of the total, which is in keeping with the known ENU mutation spectra in vitro and in vivo. The high rescue efficiency of this plasmid-based model, in combination with its sensitivity to a broad spectrum of mutations, including large deletions, makes it very suitable as a general in vivo mutagenicity test system.

Plasmid-based transgenic mouse model for studying in vivo mutations.

A new transgenic mouse model for studying in vivo somatic mutations is based on the efficient recovery of chromosomally integrated lacZ-containing plasmids, using magnetic beads.

Use of transgenic mouse models for studying somatic mutations in aging.

Theories on the causes of aging, based on the accumulation of somatic mutations in tissues of an organism, were formulated decades ago, but remain insufficiently tested. Transgenic animals, equipped with integrated bacterial reporter genes that can be efficiently rescued from total genomic DNA of all tissues and organs, represent ideal tools for investigating the types and frequencies of spontaneous mutants accumulating during aging. The first of such systems, based on the transgenic integration of bacteriophage lambda shuttle vectors that contain the bacterial lacZ gene as mutational target, was constructed in our laboratory and is now routinely used. Results obtained with this and the related LacI system that are relevant for the somatic mutation theory of aging will be discussed. One conclusion is that, due to the nature of the transgene, lambda-based systems have the disadvantage that deletion type mutations are underrepresented in comparison to point mutations. To overcome those limitations, we constructed a new transgenic mouse model carrying a pUR288 plasmid shuttle vector with the lacZ reporter gene. Some preliminary data obtained with this model serve to illustrate its potential use to extensively test the somatic mutation theory of aging.

Spontaneous and X-ray-induced deletion mutations in a LacZ plasmid-based transgenic mouse model.

Transgenic mouse mutation models carrying bacterial marker genes in bacteriophage lambda shuttle vectors have been applied to study spontaneous or induced mutations in vivo. However, due to the nature of the shuttle vector these models are insensitive to large deletions. Clastogenic agents, which predominantly induce large deletions, were therefore found to yield very low responses in these assays. Here we report the use of LacZ plasmid-based transgenic mice, allowing the detection of a broad spectrum of mutations. Treatment of mice with X-rays (5 x 50 rads) resulted in induction of up to about 5-fold higher mutation frequencies in lung, spleen and liver. Analysis of spontaneous and induced mutant LacZ genes indicated that at least 40-50% of all mutations were caused by deletions. The possibility of detecting a broad spectrum of mutations with this system suggests that the LacZ plasmid-based transgenic mouse may be the mammalian model of choice for studying spontaneous and induced mutations in vivo.

Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice.

The lacZ transgenic mouse (Muta mouse) model was used to examine the timing of ethylnitrosourea (ENU)-induced mutations in germ cells. The spectrum of mutations was also determined. Animals received five daily treatments with ENU at 50 mg/kg and were sampled at times up to 55 days after treatment. In mixed germ-cell populations isolated from seminiferous tubules, there was little increase in the mutant frequency 5 days after treatment; subsequently, there was a continuous increase until the maximum (17.5-fold above background) was reached by approximately 35 days. In the spermatozoa, an increase in mutant frequency was not seen until 20 days after treatment, with the maximum (4.3-fold above background) being achieved no sooner than approximately 35 days. Based on the timing of sampling, these data demonstrate the detection of both spermatogonial and postspermatogonial, mutations. The most prominent feature of the ENU-induced base-pair mutations in testicular germ cells sampled 55 days after treatment is that 70% are induced in A.T base pairs, compared to only 16% in spontaneous mutations. These findings are consistent with comparable data from ENU studies using assays for inherited germ-cell mutations in mice. This study has demonstrated the utility and potential of the transgenic mouse lacZ model (Muta mouse) for the detection and study of germ-cell mutations and provides guidance in the selection of simplified treatment and sampling protocols.

Sequence spectra of spontaneous lacZ gene mutations in transgenic mouse somatic and germline tissues.

As a critical step in determining whether transgenic mouse gene mutation systems are suitable models for the detection and quantification of induced gene mutations in vivo, spontaneous mutant frequencies and mutation spectra have been characterized for liver, bone marrow, and male germ cells of the lacZ transgenic mouse strain 40.6. The lacZ transgene is carried on a recombinant bacteriophage lambda shuttle vector that is recovered from mouse genomic DNA, and analysed in vitro for mutations that occurred in the mouse tissues. Mutations are detected visually as clear or pale blue plaques when X-gal is the substrate for beta-galactosidase; whereas, the wild-type plaques are dark blue. There was no statistical difference in the mutant frequency among the three tissues studied, the pooled mutant frequency being 2.23 +/- 0.41 per 10(5) pfu. The predominant type of mutation was GC-->AT transitions, with most occurring in 5'-CpG dinucleotides, suggesting that the deamination of 5-methylcytosine is the main mechanism of mutagenesis. There was, however, a statistically significant difference in the base pair substitution mutation spectrum for the liver and bone marrow when mutations were grouped according to GC or AT base-pairs. The proportion of transition versus transversion mutations was also statistically different among the three tissues, resulting mainly from the fact that germ cells were different from both bone marrow and liver. A lower number of spontaneous transitions in male germ cells was accompanied by an increase in transversions, with the proportion of GC-->AT transitions in 5'-CpG sites also declining.(ABSTRACT TRUNCATED AT 250 WORDS)

LacZ transgenic mouse models: their application in genetic toxicology.

Gene mutations have been implicated in the etiology of cancer, developmental anomalies, genetic disease and aging. Many different methods for mutation detection have been developed and applied to obtain a more fundamental insight in the chain of molecular events that ultimately lead to mutations. Most of these methods, however, can only be applied to cultured cells and therefore do not allow comparative analysis of mutations in various organs and tissues in an intact organism. The main difficulty in studying mutagenesis in chromosomal DNA is to identify and isolate mutated genes with a high efficiency. Here we describe the development and application of LacZ transgenic mouse models for studying, in different organs and tissues, spontaneous or induced mutations. Such models allow study of the induction of DNA damage, repair, mutagenesis and carcinogenesis in one animal system. Accordingly, results obtained may ultimately provide greater insight into the chain of events from in vivo exposure to genotoxic agents to mutations and their ultimate physiological endpoints. In addition to their use in fundamental research, transgenic animal mutation models find a major application in the field of genetic toxicology testing, in particular with respect to organ specificity.

DNA sequence analysis of spontaneous mutations at a LacZ transgene integrated on the mouse X chromosome.

Transgenic mice with integrated shuttle vectors containing the LacZ mutational target gene were used to study spontaneous mutational events in vivo. The transgenic mouse strain used carries the LacZ transgene on the X chromosome and was previously found to be characterized by approximately 25-fold higher spontaneous mutation frequency in liver and brain compared with at least three other transgenic mouse strains. To determine the nature of in vivo spontaneous mutational events, 35 mutant LacZ genes isolated from liver and brain of mice from strain 35.5 were analyzed at the DNA sequence level. The results obtained indicate that single base-pair changes were predominant in both liver and brain. However, in liver the majority of mutations were transitions whereas in brain transversions were predominantly observed. Six mutants appeared to contain multiple dispersed mutations, separated by as much as 44 bp. Mutations were generally located within a 500 bp region encoding the active site of the beta-galactosidase protein. Our results indicate that spontaneous mutations at the LacZ transgene are tissue specific and dependent on the chromosomal position of the LacZ transgene.

Plasmid rescue from transgenic mouse DNA using LacI repressor protein conjugated to magnetic beads.

A method for the efficient rescue of lac operator containing plasmids from transgenic mouse genomic DNA is described. The method is based on the high affinity of the LacI repressor protein for the lac operator sequence. Using the LacI repressor protein conjugated to magnetic beads, more than 95% of plasmid sequences could be purified from restriction enzyme digested genomic DNA. After circularization, the plasmids were introduced into Escherichia coli by means of electroporation. Since the plasmid was cloned into a bacteriophage lambda vector, the efficiency of plasmid rescue could easily be compared with in vitro packaging. Our results indicate that plasmid rescue is about 25 times more efficient. Application of this method should be especially useful with transgenic mouse models harboring LacZ plasmid shuttle vectors for studying spontaneous or induced mutations in vivo.

Detection of migrated allogeneic oligodendrocytes throughout the central nervous system of the galactocerebrosidase-deficient twitcher mouse.

Galactocerebrosidase-deficient oligodendrocytes of 'twitcher' (twi/twi) mice degenerate prematurely. Transplantation of normal bone marrow cells has been shown to alleviate symptoms and to prolong survival time. However, characteristic ataxia ('twitching') is not cured. In an attempt to improve further the condition of twitcher mice, allogeneic foetal liver cells were transplanted as a source of normal haemopoietic stem cells and supplemented with intracerebral transplantation of foetal brain cells. A reliable method was developed to detect donor-type cells in brain tissue. Bacteriophage lambda transgenic foetal mice were used as donors of both foetal liver and brain cells. Integrated copies of lambda DNA in donor cells were detected by in situ hybridization with biotinylated probes, which were then stained using streptavidin alkaline phosphatase. This technique was combined with immunohistochemistry to distinguish donor-type oligodendrocytes from macrophages. Immunoperoxidase staining with an antiserum to carbonic anhydrase-II produced dark perikarya of oligodendrocytes. The results demonstrated that local foetal brain cell grafts resulted in a wide dissemination of donor-type oligodendrocytes throughout the twitcher brain. The addition of a foetal brain cell graft to haemopoietic cell transplantation resulted in significantly prolonged survival of twitcher mice.

High somatic mutation frequencies in a LacZ transgene integrated on the mouse X-chromosome.

To study spontaneous and induced mutagenesis in vivo we recently constructed a series of transgenic mice harboring different numbers of bacteriophage lambda shuttle vectors, provided with a LacZ mutational target gene, integrated in their genome. The transgenic mice enabled analysis of spontaneous and induced mutation frequencies in postmitotic tissues like liver and brain. The obtained data indicated spontaneous mutation frequencies in the order of 10(-5)-10(-6). Here we report a 25-100 times higher spontaneous mutation frequency in liver and brain DNA of mice from strain 35.5, with the lambda-gt10LacZ concatemer integrated on the X-chromosome. These results indicate the presence of a mutational 'hot spot' in the mammalian somatic genome in vivo.