Sequenc spectra of spontaneous lacZ gene mutations in trransgenic mouse somatic and germline tissues

As a critical step in determining whether transgenic mouse genemutation systems are suitable models for the detection and quantificationof induced gene mutations in vivo, spontaneous mutant frequenciesand mutation spectra have been characterized for liver, bonemarrow, and male germ cells of the lacZ transgenic mouse strain40.6. The lacZ transgene is carried on a recombinant bacteriophage     

System issues: A test for neutrality of mutations of the lacZ transgene

Neutral mutations are stable alterations in DNA that are neither beneficial nor damaging to the cell. Once a mutation is fixed in the DNA, the mutant frequency should increase to its maximum and plateau thereafter, provided that enough expression time is given. Evidence has been obtained indicating neutrality for the endogenous Dlb-1 locus and for the lacl in the BigBlue™ transgenic system, and is reported here for lacZ in the Muta™Mouse system. We have found that, after an acute dose of ethylnitrosourea (ENU, 250 mg/kg) the mutant frequency for both lacZ transgene and Dlb-1 remained constant between 10 and 70 days after mutagenesis in the small intestine, where cell turnover is rapid (∼7 days). The some concept should apply to other tissues. © 1996 Wiley-Liss, Inc.     

System issues: Spontaneous mutation in Big Blue® transgenic mice: Analysis of age,gender, and tissue type

A lacl-containing transgenic mouse mutation detection system (Big Blue®) was used to determine the frequency and spectrum of spontaneous mutation in two rapidly dividing tissues (male germ cells and thymus) and one slowly dividing tissue (brain) at 3 and 10 months of age. By screening 9.4 million γ plaques, a total of 343 circular mutant plaques were recovered from the three tissues. The mutation frequencies and spectra were determined by sequencing the lacl gene and associated lacZ operator in all samples and correcting for “jackpot” mutations. The mutation frequencies and spectra were similar in all three tissues and there were no age-dependent or gender-dependent changes. When the mutation spectrum in each tissue was compared by utilizing large numbers of independent mutations (average: 75 per tissue), there was evidence for small tissue-specific differences. The spectrum of “jackpot” mutations, which clearly represents in vivo mouse-derived mutations, was similar to that of nonjackpot mutations, providing additional evidence that observed mutations occur in mouse. In the aggregate, the results suggest that there is: (i) a core mutation frequency and spectrum that is modified weakly by tissue-specific metabolism, and (ii) a steady-state level of spontaneous mutation in adult mice reflecting the balance between the accumulation of new mutations and the elimination of mutated cells by either selection against suboptimal cellular function or apoptosis triggered by accumulated DNA damage. © 1996 Wiley-Liss, Inc.     

Other transgenic mutation assays: Tissue specificity of spontaneous point mutations in λsupF transgenic mice

Transgenic mice carrying multiple copies of a recoverable λ phage shuttle vector carrying the supF mutation reporter gene (λsupF) were constructed for the purpose of studying mutagenesis in a whole animal. Spontaneous mutations in rescued supF target genes from mouse liver and skin were analyzed. The mutation frequency was similar in both tissues (in the range of 2 × 10⁻⁵), but the spectrum of point mutations was distinct, with transitions common in the skin and transversions more prominent in the liver (P = 0.01). These results may help to elucidate pathways of endogenous mutagenesis in vivo, and they illustrate potentially important tissue-specific differences in genetic instability. © 1996 Wiley-Liss, Inc.     

Spectrum of spontaneous mutations in liver tissue of lacI transgenic mice

The advent of transgenic technology has greatly facilitated the study of mutation in animals in vivo. The Big Blue® mouse system, transgenic for the lacI gene, permits not only the quantification of mutations in different tissues but also provides for the generation of in vivo-derived mutational spectra. This report details the sequence alterations of 348 spontaneous mutations recovered from the liver of 6–8-week-old male Big Blue® mice. The spectra recovered from two strains of mice, C57Bl/6 and B6C3F1, were compared and found to be very similar. The predominant mutations are G:C → A:T transitions, with 75% of these occurring at 5′-CpG-3′ sequences. This mutational bias is consistent with deamination-directed mutation at methylated cytosine bases. The second most common class of mutations is G:C → T:A transversions. A significant clonal expansion of mutants was found in several animals, and this was used to make an approximate correction of the mutant frequency such that the most conservative estimate of mutation frequency is presented. The establishment of this substantial database of spontaneous mutations in the liver of Big Blue® mice is intended to serve as a reference against which mutations recovered after treatment can be compared. Environ. Mol. Mutagen. 30:273–286, 1997 © 1997 Wiley-Liss, Inc.     

In vivo mutation in gene A of splenic lymphocytes from phiX174 transgenic mice

Single-burst analysis was applied to a forward assay for gene A mutation in splenic lymphocytes of phiX174 transgenic mice for the purpose of optimizing analytical parameters for identifying in vivo mutations. The effect of varying the cutoff value for an in vivo burst on induced mutant frequency, fold increase, and the significance of the difference between control and N-ethyl-N-nitrosourea (ENU)-treated mice was calculated by two different methods. The plating density was reduced to an average of less than 10 background mutant plaques per aliquot in order to separate in vitro bursts. The spectrum of mutations contributing < 60 plaques per aliquot from control animals was not significantly different from the control spectra from E. coli or transgenic phiX174 cells in culture. The mutant spectra from ENU-treated animals was highly different between mutant bursts of > 80 plaques per aliquot compared to mutations contributing < 60 plaques per aliquot (P < 0.000001), the former fitting the spectrum expected for ENU-induced mutations. The latter spectrum was also different from control animals and E. coli (P < 0.000001), suggesting the difference was caused by ex vivo mutation. With the mutations found in this study, the total number of reported target sites for gene A is now 33. The results support the interpretation that, in contrast to results for the lacI transgene, 100% of mutants isolated in gene A from control animals and cells were fixed in E. coli. We attribute the difference between the two genes to hot-spot sites for mutation in gene A and to a testable hypothesis that the mosaic plaque assay for the lacI transgene underestimates the frequency of ex vivo mutants.     

Further characterization and validation of gpt delta transgenic mice for quantifying somatic mutations in vivo.

The utility of any mutation assay depends on its characteristics, which are best discovered using model mutagens. To this end, we report further on the characteristics of the lambda-based gpt delta transgenic assay first described by Nohmi et al. ([1996]: Environ Mol Mutagen 28:465-470). Our studies show that the gpt transgene responds similarly to other transgenic loci, specifically lacZ and cII, after treatment with acute doses of N-ethyl-N-nitrosourea (ENU). Because genetic neutrality is an important factor in the design of treatment protocols for mutagenicity testing, as well as for valid comparisons between different tissues and treatments, a time-course study was conducted. The results indicate that the gpt transgene, like cII and lacZ, is genetically neutral in vivo. The sensitivities of the loci are also equivalent, as evidenced by spontaneous mutant frequency data and dose- response curves after acute treatment with 50, 150, or 250 mg/kg ENU. The results are interesting in light of transgenic target size and location and of host genetic background differences. Based on these studies, protocols developed for other transgenic assays should be suitable for the gpt delta. Additionally, a comparison of the gpt and an endogenous locus, Dlb-1, within the small intestine of chronically treated animals (94 microg/mL ENU in drinking water daily) shows differential accumulation of mutations at the loci during chronic exposure. The results further support the existence of preferential repair at endogenous, expressed genes relative to transgenes.     

Poly (ADP‐ribose) polymerase‐1 deficiency does not affect ethylnitrosourea mutagenicity in liver and testis of lacZ transgenic mice

Poly (ADP‐ribose) polymerase‐1 (Parp1) has been implicated in DNA base excision repair, single‐ and double‐strand break repair pathways, as well as in cell death by apoptosis or necrosis. We used Parp1^?/? lacZ plasmid‐based transgenic mice to investigate whether Parp1 deficiency influences the in vivo mutagenic and clastogenic response to the alkylating agent N‐ethyl‐N‐Nitrosourea (ENU) in somatic and germ‐cell tissues. The comparison of the lacZ mutant frequencies (MFs) between Parp1^+/+ and Parp1^?/? mice showed that the ablation of Parp1 does not affect the spontaneous or ENU‐induced MFs in liver and testis. In addition, the spectrum of the ENU‐induced mutations was not dependent on the Parp1 status, given that similar spectra, consisting mostly of point mutations and a small fraction of deletions/insertions, wereobserved in organs of both Parp1^?/? and Parp1^+/+ mice. Sequencing of point mutations revealed a consistent significant increase in A:T → T:A base substitutions, typically induced by ENU. Overall, we observed that neither the frequency nor the spectrum of ENU‐induced mutations demonstrated a specificity that could be attributed to the Parp1 impairment in mice organs. The analysis of micronucleus frequency in peripheral blood reticulocytes showed that ENU was clastogenic in both Parp1^?/? and Parp1^+/+ mice and had a strong cytotoxic effect in Parp1^?/? mice only. The present data suggest that, at a whole‐organism level, Parp1‐independent repair mechanisms may be operative in the removal of ENU‐induced DNA lesions or that highly damaged cells may be preferentially committed to death when Parp1 is inactivated. Environ. Mol. Mutagen. 2010. © 2010 Wiley‐Liss, Inc.     

Database and software for the analysis of mutations at the lacZ locus in transgenic rodents

The use of transgenic rodents is becoming increasingly widespread in genetic toxicology. In an effort to centralize and standardize the information regarding mutations in rodents bearing the lacZ transgene, we have created a computerized database that contains published information about DNA sequence alterations on over 100 mutants. Information on the literature citation, mutagenic conditions, organs from specific animals, mutation frequency in each organ, specific mutation, amino acid change, and other data are provided for each mutant. We have also produced a software package for the analysis of the lacZ database. Routines have been developed for the analysis of single base substitutions, including programs to 1) determine whether two mutational spectra are statistically different, 2) determine whether mutations show a DNA strand bias, 3) determine the frequency of transitions and transversions, 4) display the number and kind of mutations observed at each base in the coding region, 5) perform nearest-neighbor analysis, and 6) display mutable amino acids in the lacZ protein. The software runs only on IBM-compatible machines running Microsoft Windows. The software and lacZ database are freely available via the Internet ( http://sunsite.unc.edu/dnam/mainpage.html ). These programs simplify the analysis of the rapidly increasing information about lacZ mutation. The programs permit the facile comparison between different lacZ data sets as well as the identification of mutational patterns that may be of importance to experimenters studying the mechanisms of mutation and mutational spectra in transgenic animals. © 1996 Wiley-Liss, Inc.     

Regeneration of the liver in mice treated with a mixture of hepatotoxins in delayed periods after bone marrow transplantation

Possible effect of bone marrow cells on liver regeneration was studied in mice injected with a mixture of hepatotoxins (allyl alcohol and CCl₄) in a dose equal to LD₅₀. The mixture of hepatotoxins was used to minimize the restitution regeneration of the liver. The dose of allyl alcohol causing (in combination with CCl₄) maximum liver damage was selected beforehand. Increasing the dose of allyl alcohol in the two-component mixture resulted in more severe necrosis of the liver. The maximum dose of alcohol (50 mg/kg) in combination with CCl₄ caused irreversible injury to the liver leading to 100% mortality after 2–4 days. In radiation chimeras reconstituted by bone marrow cell transplantation, in which liver damage was induced by a mixture of hepatotoxins containing the maximum dose of allyl alcohol, we observed normalization of liver tissue structure and function. The mechanism of this effect is not clear. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 141, No. 4, pp. 462–465, April, 2006     

绿色荧光蛋白转基因小鼠骨髓基质细胞体外诱导分化为神经元样细胞

目的 研究绿色荧光蛋白转基因小鼠骨髓基质细胞(GFP-GM-BMSCs)在体外无血清培养基+神经细胞因子诱导条件下,向神经细胞分化的能力。方法 用贴壁法体外培养GFP-GM-BMSCs,取第3代GFP-GM-BMSCs,用含浓度均为20μg/L的表皮生长因子(EGF)和碱性成纤维细胞生长因子（bFGF）的无血清培养基（neurobasal-A+2%B27）诱导分化。第5天用免疫细胞荧光方法检测巢蛋白(nestin)的表达,第10天用神经元烯醇化酶 (NSE)、神经胶质纤维酸性蛋白(GFAP)免疫细胞化学方法鉴定阳性细胞。结果 GFP-GM-BMSCs经无血清培养基+神经细胞因子诱导后,细胞胞体变圆,伸出细长突起, 有的突起连接成网,呈神经元样形态。诱导第5天,nestin阳性表达的细胞为40.24%+5.09%;第10天,NSE阳性、GFAP阳性的细胞分别为36.43%+5.27%和49.73%+6.28%。 结论 GFP-GM-BMSCs在体外含EGF、bFGF的无血清培养基中,能分化成神经元样细胞。     

Origin and evolution of somatic cell testicular tumours in transgenic mice

Transgenic mice bearing a construct in which the expression of the SV40 oncogene is directed by the AMH promoter (AT mice) develop testicular tumours in adult life. We aimed to study early steps of tumour development and characterize tumours at different ages by histological, morphometric, and immunohistochemical techniques. One‐ to 3‐month‐old AT mice depicted multifocal Leydig cell hyperplasia. The testicular volume occupied by interstitial tissue was significantly higher in 3‐month‐old AT mice in comparison with littermate controls. Between 5 1/2 and 7 months, microscopic interstitial tumours developed that progressively evolved to form large confluent areas of high mitotic index in 7‐ to 14‐month‐old AT mice. Tumour cells had the characteristics and histoarchitecture of Leydig cells, or formed solid cord‐like structures reminiscent of those seen in Sertoli cell tumours. Hyperplastic areas and tumours diffusely expressed 3β‐hydroxysteroid dehydrogenase (3β‐HSD) in Leydig cell areas. AMH expression was negative in Leydig cell conglomerates and tumours and variable in cord‐like tumours. The SV40 T antigen and markers of cell proliferation (PCNA) were intensely positive in hyperplastic cells and tumours. Control mice of similar ages showed neither hyperplasia nor tumours, and SV40 T expression was always negative. In conclusion, transgenic mice develop large testicular tumours that are preceded by interstitial hyperplasia and microtumours. The histological and immunohistochemical phenotype of tumours (Leydig and Sertoli cell differentiation, positive 3β‐HSD, and variable AMH) suggests a mixed differentiation of somatic cells of the specialized gonadal stroma. The finding that an oncogene directed by a promoter specifically active in fetal Sertoli cells has given rise to testicular tumours of mixed differentiation is compatible with a common origin of Leydig and Sertoli cells from the specific stroma of the gonadal ridge, as supported by double labelling experiments in fetal mice showing co‐localization of the transgene with Sertoli and Leydig cell markers. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Silicotic lesions of the bone marrow: histopathology and microanalysis

Silica deposition and characteristic nodular silicotic lesions of the bone marrow, virtually unknown features of silicosis, are described in a case of severe lung silicosis with silicotic granulomas of the liver and spleen. Scanning electron microscopy and X-ray microanalysis confirmed the presence of quartz and feldspars. The bone marrow lesions included inconspicuous accumulations of silica-containing macrophages, free silica, slight lymphocyte and plasma cell infiltration, and reticulin fibre formation; and development of slightly larger partly fibrous silicotic nodules, comparable to those of the lung, liver, and spleen. Silicosis must therefore be considered in the differential diagnosis of bone marrow granulomas.     

Analysis of spontaneous and induced mutations in transgenic mice using a lambda ZAP/lacI shuttle vector.

A short term, in vivo mutagenesis assay has been developed utilizing a lacl target gene contained within a lambda ZAP shuttle vector which has been incorporated into transgenic mice. Following chemical exposure, the target gene was recovered from mouse genomic DNA by mixing the DNA with in vitro lambda phage packaging extract. Mutations within the lacl target were identified by infecting host E. coli with the packaged phage and plating on indicator plates containing Xgal. Phage plaques with mutations in the lacl appeared blue, while intact phage were colorless. The ratio of blue plaques to colorless plaques is a measure of the mutagenicity of the compound. This system was used to obtain significant induction (up to 74-fold) over background levels for a variety of compounds, including N-ethyl-N-nitrosourea, benzo(a)pyrene (BaP), cyclophosphamide, and methylnitrosourea. Sequence analysis of selected mutant clones derived from this system was accomplished through the use of partial filamentous phage origins which allow rapid transfer of the target gene from phage to plasmid. Sequence analysis of spontaneous mutants derived from the mice primarily found of base substitutions, differing markedly from the previous data for spontaneous mutations in lacl derived from E. coli, where the preponderance of mutations were found at a single site, a repeated tetramer sequence. Upon sequence analysis of BaP derived base substitutions, only transversions were obtained, consistent with the known mechanism of BaP mutagenesis. Use of the well-characterized lacl gene in transgenic mice should allow for extrapolation of the extensive pool of in vitro data to whole animals, as well as provide insight into the tissue specific effects of mutagenic compounds.     

绿色荧光蛋白转基因小鼠不同源性间充质干细胞原代培养及生物学特性比较

 目的： 探讨绿色荧光蛋白转基因小鼠脐带和骨髓源性间充质干细胞 （MSCs）的体外分离培养方法、生物学特性、表面标志及多向分化潜能。方法：应用Ⅱ型胶原酶消化培养法分离脐带MSCs及密度梯度离心法分离骨髓MSCs进行体外培养。在倒置显微镜下观察2种细胞的生长特点,运用生长曲线和MTT法检测其原代细胞增殖能力,台盼蓝法测定细胞传代成活率,采用流式细胞术测定2种第3代（P3）细胞DNA周期及表面标志物的表达,并比较其向成脂细胞和成骨细胞的分化潜能。结果：酶消化法分离培养的脐带MSCs 1 d后,细胞贴壁呈成纤维形,2 d后呈漩涡状生长且增殖明显,3 d后达80%融合即可传代;应用密度梯度离心法分离骨髓MSCs,体外培养4 d后,细胞贴壁呈圆形、梭形和多角形生长,5 d后呈克隆样生长且增殖明显,7 d后达80%融合即可传代。原代培养的脐带MSCs生长曲线近似“S”形,骨髓MSCs 生长曲线较平缓;MTT法显示脐带MSCs在3～5 d增殖较明显,骨髓MSCs 7 d后细胞增殖较明显。2种P3细胞传代成活率均为96%以上,G0/G1期细胞均为85%以上,无明显差异（P>0.05）;2种P3细胞CD44、CD90和CD105阳性率均为(60.7±2.3)%以上高表达,CD45、CD19、CD14和CD79a均为(25.6±4.8)%低表达,两者无明显差异（P>0.05）;2种MSCs在体外均具有向成骨细胞和成脂细胞分化的潜能,脐带MSCs向成骨及成脂细胞分化率均为90%以上,与骨髓MSCs的分化潜能比较有显著差异（P<0.05）。结论：脐带MSCs较骨髓MSCs具有较强的增殖能力及分化潜能。绿色荧光蛋白转基因小鼠的脐带MSCs可作为较好干细胞示踪的细胞源。     

Identification of the novel allele HLA-B*0809 in a Caucasian individual: estimation of allogeneic potential between B*08 variants

The identification of the new allele HLA-B*0809, which was found in a Caucasian individual, is described. In the sequence analysis the new allele differs from B*0801 by six nucleotides located in exon 3. As the structure is identical to a variety of other HLA-B alleles, it is likely that the new allele originated by gene conversion. At the protein level, the new allele has two amino acid differences compared to B*0801. Comparing the residues of the alpha1 and alpha2 domains of the B*08 variants to each other, a high degree of polymorphism was found. Three alleles differ from B*0801 by only one amino acid residue whereas the other comparisons revealed at least two disparities. B*0809 differs from the other B*08 variants by at least two amino acid residues, suggesting that mismatching may provoke alloreactivity and thus impair clinical outcome of bone marrow transplantation. Among the B*08 alleles with a single amino acid difference, the mismatch combinations B*0801 vs. B*0805 and B*0801 vs. B*0807 appear to be the most compatible based on structural data.     

Expression of bone marrow stromal cell specific antigen during murine development:Its expression in embryonic hematopoietic tissues as well as in other developing tissues

Monoclonal antibody R4-A9 demonstrated specificity for a cell surface antigen of stromal cells in murine bone marrow and spleen. In order to Identify patterns of expression that may elucidate the potential role of R4-A9 antigen, the developmental expression of this antigen in mouse embryos from 8 days post-coitum to 5 days post-partum was investigated by immunohistochemistry. At an early developmental stage, weak staining for R4-A9 antigen could be detected in the yolk sac. At later stages, strong staining of this antigen was detected predominantly in the embryonic liver, the main site of embryonic hematopoiesis. However, concomitant with the decreased staining in the liver, increased expression of this antigen was observed in bone marrow and spleen. Therefore, the changes in expression in those hematopofetic tissues suggest that its expression is coordinately regulated during the developmental stage of the sites of embryonic hematopoiesis. Compared with the distribution of R4-A9 antigen in adult tissues as previously reported, the expression of this antigen in fetal tissues was more widespread during the period of organogenesis, and was most abundant in other developing tissues, including the heart, skin, and lung. In contrast, fetal expression detected in hematopoietic and other developing tissues was lost after birth. These results taken together show a marked gradient of R4-A9 antigen expression, with the highest level at the peak of organ development, raising the possibility that this molecule may act as a growth/differentiation factor both in hematopoietic and other developing tissues in a fetus.     

Ultrastructural characteristics of bone marrow in patients with hematological disease: a study of 13 cases

There are few transmission electron microscopic studies on bone marrow biopsies of patients with hematological disease owing to the difficulty of overcoming the artifacts of decalcification. Following the fixation of bone marrow biopsies thoroughly before a mild decalcification procedure, ultrastructural studies were performed on 13 patients with varied hematological diseases. Notable features included blood cell disorganization, fibroblast activation, myofibroblast transformation, as well as accumulation of collagen and extracellular amorphous matrix. In addition, excessive blood cell death in leukemia, apoptosis, and macrophage phagocytosis in myelodysplastic syndrome and polycythemia vera, as well as degranulation of eosinophils and megakaryocytes in chronic idiopathic myelofibrosis were predominant, respectively. The observations suggest that polyclonal fibroblast proliferation and extracellular matrix accumulation may result from inflammation resulting from excessive cell death and active material release of blood cells in the bone marrow of patients with hematological disease.