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Kidney diseases lead to the failure of urinary excretion of metabolism products. In the Munich ethylnitrosourea (ENU) mouse mutagenesis project, which is done on a C3H inbred genetic background, blood samples of more than 15,000 G1 offspring and 500 G3 pedigrees were screened for alterations in clinical-chemical parameters. We identified 44 animals consistently exhibiting increased plasma urea concentrations. Transmission analysis of the altered phenotype of 23 mice to subsequent generations led to the establishment of five mutant lines. Both sexes were affected in these lines. Urinary urea levels were decreased in the mutants. In addition, most mutants showed increased plasma and decreased urinary creatinine levels. Pathological investigation of kidneys from the five mutant lines revealed a broad spectrum of alterations, ranging from no macroscopic and light microscopic kidney alterations to decreased kidney weight-to-body weight ratio, dilation of the renal pelvis, and severe glomerular lesions. Thus screening for elevated plasma urea levels in a large-scale ENU mouse mutagenesis project resulted in the successful establishment of mouse strains which are valuable tools for molecular studies of mechanisms involved in urea excretion or which represent interesting models for kidney diseases.

Endogenous retroviral sequences in the pig genome represent a potential infectious risk in xenotransplantation. Porcine endogenous retrovirus (PERV) gamma sequences described to date have been classified into several families. The known infectious, human-tropic PERVs have been assigned to the PERV gamma1 subfamilies A, B and C. High copy numbers and full-length clones have also been observed for an additional family, designated PERV gamma2. The aim of this study was to examine the PERV gamma2 family by analysis of retroviral pro/pol gene sequences. The proviral load was observed to be similar among various pig breeds. Although clones harbouring an open reading frame in the examined region were found, analysis of published large PERV gamma2 clones revealed multiple deleterious mutations in each of the retroviral genes. Various recombination events between gamma2 genomes were revealed. In contrast to PERV gamma1, phylogenetic analyses did not distinguish defined subfamilies, but indicated the independent evolution of the proviruses after a single event of retroviral amplification. Expression analysis showed large PERV gamma2 transcripts and variable transcription in several tissues. Analysis of the two published gamma2 env gene sequences observed the partial lack of the receptor-binding domain. Overall, this study indicated the low infectious potential for PERV gamma2.

Mice are important models for biomedical research because of the possibility of standardizing genetic background and environmental conditions, which both affect phenotypic variability. Inbred mouse strains as well as F1 hybrid mice are routinely used as genetically defined animal models; however, only a few studies investigated the variance of phenotypic parameters in inbred versus F1 hybrid mice and the potential interference of the genetic background with different housing conditions. Thus, we analyzed the ranges of clinical chemical and hematologic parameters in C3H and C57BL/6 inbred mice and their reciprocal F1 hybrids (B6C3F1, C3B6F1) in two different mouse facilities. Two thirds of the blood parameters examined in the same strain differed between the facilities for both the inbred strains and the F1 hybrid lines. The relation of the values between inbred and F1 hybrid mice was also affected by the facility. The variance of blood parameters in F1 hybrid mice compared with their parental inbred strains was inconsistent in one facility but generally smaller in the other facility. A subsequent study of F1 hybrid animals derived from the parental strains C3H and BALB/c, which was done in the latter housing unit, detected no general difference in the variance of blood parameters between F1 hybrid and inbred mice. Our study clearly demonstrates the possibility of major interactions between genotype and environment regarding the variance of clinical chemical and hematologic parameters.

Prevention of cross-species infection with porcine endogenous retroviruses (PERV) is crucial for xenotransplantation. Previous studies described the potential risk of infection for the PERV gamma1 subfamilies A, B and C. Replication competent PERV gamma1 proviruses designated to a particular subfamily and hybrid viruses originating from retroviral recombination events between the subfamilies were observed. Future pig genome sequencing projects will reveal multiple novel PERV proviruses from additional breeds and animals. Evaluation of these viral genomes has to be carried out to assess the potential risk of retroviral cross-species infection. In this study, we tested common sequence comparison methods for the classification of PERV sequences and the detection of hybrid clones. The examination of the polymorphic nucleotide positions was found to be the most suitable procedure. We describe a fast and simple method using bioinformatic software tools which can also be applied to analogous analyses of other viral genomes.

Background/aim: Phenotype-driven screening of a great pool of randomly mutant mice and subsequent selection of animals showing symptoms equivalent to human kidney diseases may result in the generation of novel suitable models for the study of the pathomechanisms and the identification of genes involved in kidney dysfunction., Methods: We carried out a large-scale analysis of ethylnitrosourea (ENU)-induced mouse mutants for albuminuria by using qualitative SDS-polyacrylamide gel electrophoresis., Results: The primary albuminuria screen preceded the comprehensive phenotypic mutation analysis in a part of the mice of the Munich ENU project to avoid loss of mutant animals as a consequence of prolonged suffering from severe nephropathy. The primary screen detected six confirmed phenotypic variants in 2,011 G1 animals screened for dominant mutations and no variant in 48 G3 pedigrees screened for recessive mutations. Further breeding experiments resulted in two lines showing a low phenotypic penetrance of albuminuria. The secondary albuminuria screen was carried out in mutant lines which were established in the Munich ENU project without preceding primary albuminuria analysis. Two lines showing increased plasma urea levels were chosen to clarify if severe kidney lesions are involved in the abnormal phenotype. This analysis revealed severe albuminuria in mice which are affected by a recessive mutation leading to increased plasma urea and cholesterol levels., Conclusion: Thus, the phenotypic selection of ENU-induced mutants according to the parameter proteinuria in principle demonstrates the feasibility to identify nephropathy phenotypes in ENU-mutagenized mice.

Hypercholesterolemia is caused by multiple environmental factors and genetic predispositions, and plays an important role in the development and pathogenesis of various human diseases. In this study, we aimed to establish randomly mutant mouse lines showing hypercholesterolemia for their further use in the detection of novel causative alleles. In the Munich ENU Mouse Mutagenesis Project, clinical chemistry blood analysis was performed on more than 15,000 G1 mice and 230 G3 pedigrees of chemically mutagenized mice to detect dominant and recessive mutations leading to an increased plasma total cholesterol level. Using inbred C3HeB/FeJ mice we identified more than 100 animals consistently showing hypercholesterolemia. Transmission of the altered phenotype to the subsequent generations led to the production of nine hypercholesterolemic lines. A single line showed further obvious deviations in the analysis of additional clinical chemistry blood parameters. Thus, the lines produced will contribute to the search for alleles that selectively cause primary hypercholesterolemia.

Endogenous retrovirus (ERV) sequences have been found in all mammals. In vitro and in vivo experiments revealed ERV activation and cross-species infection in several species. Sheep (Ovis aries) are used for various biotechnological purposes; however, they have not yet been comprehensively screened for ERV sequences. Therefore, the aim of the study was to classify the ERV sequences in the ovine genome (OERV) by analyzing the retroviral pro-pol sequences. Three OERV beta families and nine OERV gamma families were revealed. Novel open reading frames (ORF) in the amplified proviral fragment were found in one OERV beta family and two OERV gamma families. Hybrid OERV produced by putative recombination events were not detected. Quantitative analysis of the OERV sequences in the ovine genome revealed no relevant variations in the endogenous retroviral loads of different breeds. Expression analysis of different tissues from fetal and pregnant sheep detected mRNA from both gammaretrovirus families, showing ORF fragments. Thus, the release of retroviruses from sheep cells cannot be excluded.

Prevention of cross-species infection of porcine endogenous retroviruses (PERV) is crucial for xenotransplantation. The potential risk of infection is caused by replication-competent PERV as well as by hybrid viruses derived from recombination events of distinct PERV genomes. Recently, human-tropic, replication-competent PERV genomes obtaining hybrid sequences have been observed. Here, complete polymorphism pattern analysis was performed on the full-length PERV gamma1 clones and on the complete envelope (env) gene sequences published to date. Several recombined full-length clones and a high number of different recombination patterns in the env gene were identified. In addition, recombinations with retroviral genomes not yet known were found. Thus, the potential risk of infection also exists for recombination products, including defective PERV loci.

Endogenous retroviral sequences in the pig genome (PERV) represent a potential infectious risk in xenotransplantation. All known infectious PERV have been asssigned to the PERV gamma1 family, consisting of the subfamilies A, B, and C. The aim of the study was the concise examination of PERV gamma by the analysis of the retroviral pro-pol sequences. The analysis of 52 pro-pol clones amplified in this study revealed eight PERV gamma families. In addition to four already-described families (gamma1, gamma4, gamma5, gamma6), four novel families (gamma7, gamma8, gamma9, gamma10) were identified. Quantitative analysis of the novel PERV gamma sequences in selected breeds revealed variations in the endogenous retroviral load. Open reading frames (ORF) in the amplified proviral fragment were only found for PERV gamma1. In addition, novel ORF-containing PERV gamma1 clones consisting of hybrid sequences were revealed. Sequence comparison from published full-length PERV gamma1 clones of the PERV subfamilies A, B, and C resulted in a lack of strict correlation of the classification of pro-pol and env. The results indicated the occurrence of causative recombination events between retroviral genomes. Thus, our study on PERV gamma provides new data for the evaluation and selection of pigs intended to be used in xenotransplantation.

Phenotypic and molecular genetic examinations of a transgenic mouse line showing developmental defects caused by a recessive insertional mutation were carried out. The mutant phenotype is characterized by general retardation of postnatal body growth and by the appearance of increased incisor length in the upper and lower jaw. The mutation causing the aberrant phenotype was mapped to Chromosome 13, 40 cM. Examination of the expression of the candidate genes did not show any alterations. This mutant mouse line provides a reproducible model for the identification and examination of gene(s) involved in growth and in the craniofacial development, including that of the jaws and teeth.

Ocular melanin regulates retinal development, including cell density gradients in the central retina, a region essential for normal visual acuity. In albinos this region is underdeveloped and peak cell numbers are reduced. It is not known whether there is a dosage relationship between pigmentation and the degree of this underdevelopment, as studies of the retinal effects of albinism have commonly used rodents. These have poorly developed central regions even in the wild type. Rabbits, however, have a unique, highly specialized, visual streak in the central retina where cell density gradients are very steep and these are reduced in albinos. Here, cell densities in the ganglion cell layer of separate groups of rabbits, with different levels of ocular pigmentation and known mutations of the tyrosinase gene coding sequence, were examined. These revealed reductions in peak cell densities and/or in the regions over which high cell densities were maintained in all hypopigmented phenotypes. There was no dosage relationship between levels of pigmentation and deficits in the ganglion cell layer as animals with relatively small reductions in retinal pigment had deficits comparable to those found in albinos. The greatest variability between pigmentation phenotypes was between the two completely unpigmented strains. Consequently, although pigment may regulate the development of the central retina, this study failed to show that it does so in a dose-dependent manner.

Non-insulin-dependent diabetes mellitus (type 2 diabetes) is known to be a polygenic and polyfactorial disorder. Here we describe the long-term examination of a transgenic mouse line showing the disruption of the leptin receptor (Lepr, Ob-R) gene caused by transgene insertion. The absence of the expression of the long isoform Ob-Rb uncovered a strong variation of the obesity and diabetes phenotype in the homozygous mutant mice of the outbred strain used. One part of the homozygous mice developed severe persistent early-onset obesity, whereas the other part developed cachexia after having shown initial obesity in the examination period up to 26 weeks p.p. The leptin-receptor-defective mice of this line might serve as a model for the investigation of genes modulating the development and mode of expression of diabetes., (Copyright 2000 Academic Press.)

Examination of random insertional mutations in transgenic animals harbouring an abnormal phenotype contributes to the discovery of new genes and/or the understanding of already known genes. Here we describe a transgenic mouse line showing early-onset obesity as consequence of the transgene insertion. Molecular genetic analysis revealed a partial deletion of the leptin receptor (Lepr, Ob-R) gene including the coding sequences downstream of exon 17'. This defect prevents the expression of all described membrane-bound isoforms of Ob-R except for isoform Ob-Rc in the homozygous transgenic animals. Thus, this mouse model might be useful for the investigation of the function of the short Ob-R isoforms., (Copyright 2000 Academic Press.)

Porcine primordial germ cell (PGC) derived cell lines of WAPhGH-transgenic pigs have been established that were able to contribute to chimeras. PGCs were isolated from day 25 to 28 genital ridges of more than 30 individual transgenic fetuses in order to have an easy to follow marker gene. To support undifferentiated growth, cell lines were derived and stable maintained on STO no. 8 feeder cells, a murine embryonic fibroblast cell line expressing recombinant, membrane-bound porcine stem cell factor (SCF). Fifteen lines proliferated in an undifferentiated state up to passage 13; two lines were maintained for more than 23 passages. Cell staining experiments for differentiation markers in several cell lines, indicated the presence of pluripotent cells in prolonged cultures. Further characterization using karyotyping revealed a normal, euploid set of chromosomes in cells of passages 15 and higher. Pluripotency of freshly isolated, short-term (up to 24 hr before injection) and long-term cultured, frozen/thawed cells was tested by injection into day 6 recipient blastocysts to give rise to chimeric piglets. The injected embryos (n = 209) were endoscopically transferred into the uterine horns of 11 recipient gilts. Tissue analysis from 49 fetuses and eighteen liveborn piglets for PGC contribution in chimeras was carried out using PCR analysis for the presence of the marker transgene. Thirty-two fetuses showed detectable chimerism in up to five out of 12 tissues analyzed. Skin samples from eight piglets were positive for the transgene, four of them displayed coat colour chimerism., (Copyright 1999 Wiley-Liss, Inc.)

MB/BacT, BACTEC 460 TB, and Löwenstein-Jensen (LJ) medium were evaluated in parallel for recovery of mycobacteria from 3,700 continuous clinical specimens in a routine laboratory. Mycobacteria were identified from 123 (3.3%) specimens. The recovery rates for all mycobacteria by the different systems were 91.0, 73.0, and 53.6% for BACTEC 460 TB, MB/BacT, and LJ medium, respectively. The recovery rates for Mycobacterium tuberculosis complex were 97.1, 80. 2, and 67.6%, respectively. The lack of sensitivity of the MB/BacT system was more pronounced with smear-negative specimens and resulted in a failure to detect three patients with infectious tuberculosis.

Gene-farming techniques provide an effective tool for the production of recombinant proteins in livestock. Transgenes consisting of genomic DNA sequences are as a rule more efficiently expressed than those in which the product of interest is encoded by a cDNA. However, the processing of pre-mRNA from genomic constructs may yield unexpected messenger RNAs and subsequently protein variants. We describe the appearance of different alternative mRNA splice patterns of a gene construct in which a mutant human growth hormone (hGH-N) gene is transcriptionally controlled by 2.5 kb of mouse whey acidic protein (WAP2) regulatory sequences in the mammary gland of different livestock species. Compared to the transcription products in transgenic mice harboring the same gene construct and to cell transfection experiments, expression analysis in transgenic pigs and rabbits revealed different mRNA splice patterns with regard to the proportion of the processed transcripts. Apart from already-known physiological mRNA splice products, previously undescribed processed hGH transcripts were observed in these species. Sequence analysis of the transgenes suggests that the species-specific hGH mRNA patterns may be caused by species- and tissue-specific differences in trans-acting splice factors., (Copyright 1999 Academic Press.)

Transgenic mice provide a valuable tool in all fields of basic and applied biological and medical research. In this study, we describe the fate of integrated transgenes in the mammalian host genome over a large number of generations. The stability of the germ-line transmission of integrated tyrosinase transgene copies was monitored up to generation F20 in a large number of individuals from seven transgenic mouse lines. Phenotypic and molecular genetic analysis of the offspring both within the different lines and in cross-breeding experiments revealed the high stability of the transgene integration sites in mice. Only very few individuals were affected by a transgene copy loss. These results indicate that, once homozygous transgenic lines are established, breeding programs can be continued to a high number of generations without further stringent molecular genetic analysis.

The tyrosinase gene is known to be essential for melanization and has been shown to rescue pigmentation in albino mice. Previously we have described the strict copy-number-dependent expression of a murine wild-type tyrosinase gene construct over several generations in transgenic mice. In this study, we analysed the same gene construct as a marker gene for the transmission and expression of transgenes in rabbits. Using an albino hybrid strain, we produced transgenic rabbits expressing the murine tyrosinase gene. Strict correlation between integration and expression of the transgene and stable germline transmission of the integrated gene construct according to the Mendelian pattern of inheritance was observed. Thus, breeding control was facilitated by simple phenotypic examination of the transgenic animals. In contrast to mice transgenic for the same gene construct, tyrosinase-transgenic rabbits showed a greater variety in hue, intensity and extent of coat pigmentation, which is caused by the diversity in the loci affecting the melanization. Benefits and limitations of tyrosinase as a marker gene for the detection of homozygous individuals in the albino hybrid strain used are discussed.

The generation of transgenic mice with mammalian genes cloned in yeast artificial chromosomes (YACs) has generated great interest in the field of gene transfer into livestock. Many of the problems associated with standard transgenesis-such as lack of crucial regulator elements and position effects related to the integration site, which lead to variation in expression levels irrespective of the dose of the transgene-have been practically overcome. The large size of YAC-derived gene constructs (in excess of 1 Mb) facilitates the presence and transfer of all elements required for the faithful regulation of a gene. With the experiments discussed in this report, we have addressed the possibility of applying the obvious advantages of YAC transgenesis to farm animals. We have generated transgenic rabbits carrying a 250 kb YAC covering the mouse tyrosinase gene by pronuclear microinjection, and thus rescued the albino phenotype of the transgenic individuals. To date, this is the first demonstration of a successful transfer of large genetic units into the germ line of farm animals. This development might improve the occurrence of transgene expression at physiological levels and specific sites in livestock. YAC transgenesis therefore will be applied in genetic engineering, for example, in the production of pharmacologically interesting proteins encoded by large gene units and generating transgenic donors for xenotransplantation.

Thiols have been shown to be related to insulin secretion and to uptake of glucose into tissues. In the present study the effects of i.v. administration of acetylcysteine (N-acetylcysteine, NAC, CAS 616-91-1) on glucose consumption and plasma free thiols were studied in young healthy volunteers during hyperglycemic clamp. NAC (0.5-2.0 mg/kg) significantly increased glucose consumption. This effect was not obvious at higher doses of NAC. Plasma free NAC depended on the dose of NAC injected. The t1/2 of NAC was 11 min. NAC produced significant increases of plasma cysteine concentrations, and a slight but insignificantly increase of plasma glutathione. These data suggest that a moderate increase in plasma thiols augments glucose consumption during hyperglycemic clamp.