Monitoring of Venus transgenic cell migration during pregnancy in non-transgenic rabbits.

Cell transfer between mother and fetus were demonstrated previously in several species which possess haemochorial placenta (e.g. in humans, mice, rats, etc.). Here we report the assessment of fetal and maternal microchimerism in non-transgenic (non-TG) New Zealand white rabbits which were pregnant with transgenic (TG) fetuses and in non-TG newborns of TG does. The TG construct, including the Venus fluorophore cDNA driven by a ubiquitous cytomegalovirus enhancer, chicken ß-actin promoter (CAGGS), was previously integrated into the rabbit genome by Sleeping Beauty transposon system. Three different methods [fluorescence microscopy, flow cytometry and quantitative polymerase chain reaction (QPCR)] were employed to search for TG cells and gene products in blood and other tissues of non-TG rabbits. Venus positive peripheral blood mononuclear cells (PBMCs) were not detected in the blood of non-TG littermates or non-TG does by flow cytometry. Tissue samples (liver, kidney, skeletal and heart muscle) also proved to be Venus negative examined with fluorescence microscopy, while histology sections and PBMCs of TG rabbits showed robust Venus protein expression. In case of genomic DNA (gDNA) sourced from tissue samples of non-TG rabbits, CAGGS promoter-specific fragments could not be amplified by QPCR. Our data showed the lack of detectable cell transfer between TG and non-TG rabbits during gestation.

On the emerging role of rabbit as human disease model and the instrumental role of novel transgenic tools.

The laboratory rabbit (Oryctolagus cuniculus) is widely used as a model for human diseases, because of its size, which permits non-lethal monitoring of physiological changes and similar disease characteristics. Novel transgenic tools such as, the zinc finger nuclease method and the sleeping beauty transposon mediated or BAC transgenesis were recently adapted to the laboratory rabbit and opened new opportunities in precise tissue and developmental stage specific gene expression/silencing, coupled with increased transgenic efficiencies. Many facets of human development and diseases cannot be investigated in rodents. This is especially true for early prenatal development, its long-lasting effects on health and complex disorders, and some economically important diseases such as atherosclerosis or cardiovascular diseases. The first transgenic rabbits models of arrhythmogenesis mimic human cardiac diseases much better than transgenic mice and hereby underline the importance of non-mouse models. Another emerging field is epigenetic reprogramming and pathogenic mechanisms in diabetic pregnancy, where rabbit models are indispensable. Beyond that rabbit is used for decades as major source of polyclonal antibodies and recently in monoclonal antibody production. Alteration of its genome to increase the efficiency and value of the antibodies by humanization of the immunoglobulin genes, or by increasing the expression of a special receptor (Fc receptor) that augments humoral immune response is a current demand.

Transcription specificity of the class Ib genes SLA-6, SLA-7 and SLA-8 of the swine major histocompatibility complex and comparison with class Ia genes.

Our aim was to analyse the transcription levels of the three non-classical class Ib genes SLA-6, SLA-7 and SLA-8 of the swine major histocompatibility complex in various tissues and conditions and to compare them to the transcription levels of classical class Ia genes. Twenty-five adult tissues from two pig breeds, pig renal PK15 cells infected with the Pseudorabies virus, and peripheral blood mononuclear cells (PBMCs) stimulated by lipopolysaccharide or a mixture of phorbol myristate acetate and ionomycin were included in our study. Relative transcription was quantified by quantitative real-time PCR. On average, in adult tissues and PBMCs and compared to SLA-6, the transcription level of SLA-Ia genes was 100-1000 times higher, the level of SLA-8 was 10-20 times higher, and that of SLA-7 was five times higher. Thus, SLA-8 is the most transcribed SLA-Ib gene, followed by the SLA-7 and SLA-6 genes. The highest transcription levels of SLA-Ib transcripts were found in the lymphoid organs, followed by the lung and the digestive tract. The tissue variability of expression levels was widest for the SLA-6 gene, with a 1:32 ratio between the lowest and highest levels in contrast to a 1:12 ratio for the SLA-7 and SLA-8 genes and a 1:16 ratio for the SLA-Ia genes. During PK-15 infection and PBMC stimulation, SLA-Ia and SLA-8 genes were downregulated, whereas SLA-6 and SLA-7 were upregulated, downregulated or not significantly modified. Our overall results confirm the tissue-wide transcription of the three SLA-Ib genes and suggest that they have complementary roles.

Gene affecting superoxide dismutase activity linked to the histocompatibility complex in H-2 congenic mice.

The activity of cyanide-sensitive, Cu-Zn superoxide dismutase (SOD) was studied in liver sytosols from H-2 congenic strains of mice. Higher SOD activity was found in livers of mice having H-2b/A.BY, B10, and C3H.SW/haplotypes than in those of H-2a, H-2k and H-2d haplotypes. Segregation studies supported these correlations. In H-2 recombinant strains of mice, the genes influencing the liver SOD activity occur, as ascertained by mapping techniques, at or near the H-2d region of the major histocompatibility complex.

GFP transgenic animals in biomedical research: a review of potential disadvantages.

Green Fluorescent protein (GFP) transgenic animals are accepted tools for studying various physiological processes, including organ development and cell migration. However, several in vivo studies claimed that GFP may impair transgenic animals' health. Glomerulosclerosis was observed in transgenic mice and rabbits with ubiquitous reporter protein expression. Heart-specific GFP expression evoked dilated cardiomyopathy and altered cardiac function in transgenic mouse and zebrafish lines, respectively. Moreover, growth retardation and increased axon swelling were observed in GFP and yellow fluorescent protein (YFP) transgenic mice, respectively. This review will focus on the potential drawbacks of the applications of GFP transgenic animals in biomedical research.

Identification of positive and negative regulatory regions controlling expression of the cartilage matrix protein gene.

A complex pattern of regulation of the cartilage matrix protein gene was revealed by transient expression experiments. A minimal promoter from positions -15 to +64 functioned in chondrocytes and fibroblasts. An enhancer located in the first intron exerted chondrocyte-specific stimulation on the minimal promoter activity. The same fragment, however, had a negative effect in fibroblasts. Between -334 and -15, a silencer was found which inhibited the gene expression driven from its homologous as well as heterologous promoters both in chondrocytes and fibroblasts. Additional positive and negative control regions were mapped further upstream of the promoter.

Normal skeletal development of mice lacking matrilin 1: redundant function of matrilins in cartilage?

Matrilin 1, or cartilage matrix protein, is a member of a novel family of extracellular matrix proteins. To date, four members of the family have been identified, but their biological role is unknown. Matrilin 1 and matrilin 3 are expressed in cartilage, while matrilin 2 and matrilin 4 are present in many tissues. Here we describe the generation and analysis of mice carrying a null mutation in the Crtm gene encoding matrilin 1. Anatomical and histological studies demonstrated normal development of homozygous mutant mice. Northern blot and biochemical analyses show no compensatory up-regulation of matrilin 2 or 3 in the cartilage of knockout mice. Although matrilin 1 interacts with the collagen II and aggrecan networks of cartilage, suggesting that it may play a role in cartilage tissue organization, studies of collagen extractability indicated that collagen fibril maturation and covalent cross-linking were unaffected by the absence of matrilin 1. Ultrastructural analysis did not reveal any abnormalities of matrix organization. These data suggest that matrilin 1 is not critically required for cartilage structure and function and that matrilin 1 and matrilin 3 may have functionally redundant roles.

Tumorigenicity in nude mice of dexamethasone-sensitive and -resistant, differentiated and dedifferentiated hepatoma cells.

The tumorigenicity of cell clones of the same histogenetic origin but with different dexamethasone sensitivities and states of differentiation was examined. Neither the degree of differentiation nor the glucocorticoid resistance influenced the tumor-forming capacity of Reuber rat hepatoma clones in nude mice. However, the tumorigenicity of independently isolated resistant clones maintained in vitro continuously for more than 1 year in the presence of a high concentration of dexamethasone decreased considerably. The fact that not only the differentiated but also the partially dedifferentiated and the dedifferentiated hepatoma cells grew in the form of tumors in nude mice made it possible to examine whether reexpression of the extinguished liver-specific functions occurs in the tumors. Reexpression of different liver-specific functions of the tumor cell lines derived from a partially dedifferentiated, dexamethasone-resistant clone was found, showing that in vivo tumor formation may induce differentiation.

The potential impact of new generation transgenic methods on creating rabbit models of cardiac diseases.

Since the creation of the first transgenic rabbit thirty years ago, pronuclear microinjection remained the single applied method and resulted in numerous important rabbit models of human diseases, including cardiac deficiencies, albeit with low efficiency. For additive transgenesis a novel transposon mediated method, e.g., the Sleeping Beauty transgenesis, increased the efficiency, and its application to create cardiac disease models is expected in the near future. The targeted genome engineering nuclease family, e.g., the zink finger nuclease (ZFN), the transcription activator-like effector nuclease (TALEN) and the newest, clustered regularly interspaced short palindromic repeats (CRISPR) with the CRISPR associated effector protein (CAS), revolutionized the non-mouse transgenesis. The latest gene-targeting technology, the CRISPR/CAS system, was proven to be efficient in rabbit to create multi-gene knockout models. In the future, the number of tailor-made rabbit models produced with one of the above mentioned methods is expected to exponentially increase and to provide adequate models of heart diseases.

Recombinant human tissue non-specific alkaline phosphatase successfully counteracts lipopolysaccharide induced sepsis in mice.

Sepsis is a life threatening condition that arises when the body's response to an infection injures its own tissues and organs. Sepsis can lead to shock, multiple organ failure and death especially if not recognized early and treated promptly. Molecular mechanisms underlying the systemic inflammatory response syndrome associated with sepsis are still not completely defined and most therapies developed to target the acute inflammatory component of the disease are insufficient. In this study we investigated a possibility of combating sepsis in a mouse model by intravenous treatment with recombinant human tissue non-specific alkaline phosphatase (rhTNAP) derived from transgenic rabbit milk. We induced sepsis in mice by intraperitoneal injection of LPS and three hours later treated experimental group of mice by intravenous injection with rhTNAP derived from transgenic rabbits. Such treatment was proved to be physiologically effective in this model, as administration of recombinant rhTNAP successfully combated the decrease in body temperature and resulted in increased survival of mice (80 % vs. 30 % in a control group). In a control experiment, also the administration of bovine intestinal alkaline phosphatase by intravenous injection proved to be effective in increasing survival of mice treated with LPS. Altogether, present work demonstrates the redeeming effect of the recombinant tissue non-specific AP derived from milk of genetically modified rabbits in combating sepsis induced by LPS.

Sequence, structure and chromosomal localization of Crtm gene encoding mouse cartilage matrix protein and its exclusion as a candidate for murine achondroplasia.

The mouse cartilage matrix protein gene (Crtm) was isolated from a cosmid library using a mouse Crtm cDNA fragment as probe. Crtm spans 12.2 kb from the start of translation to the polyadenylation signal sequence and comprises eight exons. Sequencing of the 1.9 kb 5' flanking region revealed a TATA-like box 72 bp upstream from the initiator Met codon as well as several cis-acting motifs known to bind eukaryotic transcription factors. Analysis of the exon-intron junctions demonstrated that the last intron does not follow the gt/ag rule but belongs to the minor class of pre-mRNA introns that contain "at" and "ac" at their 5'and 3' ends, respectively. Single-strand conformation polymorphism analysis was used to map Crtm to the distal part of chromosome 4 between the microsatellite markers D4Mit16 and D4Mit339. Achodroplasia (cn), a recessive skeletal disorder in mice, has already been mapped to this region. Immunostaining for CMP and sequence of Crtm in cn/cn mice failed to reveal any disease-specific mutations, suggesting that mutations in Crtm do not cause achondroplasia.

The zonal expression of chicken cartilage matrix protein gene in the developing skeleton of transgenic mice.

Cartilage matrix protein (CMP) is a major noncollagenous glycoprotein of hyaline cartilage with a molecular mass of about 148 kDa. It has been proposed to be involved in matrix organization by its interactions with proteoglycan and type II collagen. The 54-kDa monomers form homotrimers stabilized by disulfide bonds. The gene for chicken cartilage matrix protein was isolated, and its regulation has been studied recently in transient expression experiments. To learn more about the spatial and temporal expression of the gene during ontogenic development, we created transgenic mice via microinjection of a 21.8-kb genomic fragment, encoding the chicken cartilage matrix protein. None of the founder animals exhibited any abnormal phenotype. The developmental stage-specific expression of the transgene was examined by immunostaining with a chicken CMP specific antiserum at different stages of embryonic development in cartilage from different sources: lower and upper limb, vertebrae, ribs and nasal septum. The level of transgene expression showed marked differences in various zones of cartilage. Briefly, high levels were found in the zones of proliferating chondrocytes, while little if any transgene product was detected in the very early and hypertrophic stage of chondrogenesis. The expression pattern of the transgene correlated with the endogenous mouse CMP and did not cause any morphological changes detectable by microscopic analysis of cartilage. These data indicate that the injected CMP gene with its flanking sequences contained all the information necessary for cell type-specific expression in transgenic mice.

Stage-and tissue-specific expression of a Col2a1-Cre fusion gene in transgenic mice.

To achieve chondrocyte-specific deletion of floxed genes we generated a transgenic mouse line expressing the Cre recombinase under the control of the mouse type II collagen gene (Col2a1) regulatory regions. Northern and in situ hybridization analyses demonstrated the expression of the transgene (Col2a1-Cre) in cartilaginous tissues. To test the excision efficiency of Cre, the Col2a1-Cre strain was crossed with the ROSA26 reporter strain. LacZ staining of double transgenic mice revealed Cre activity in both chondrogenic and non-chondrogenic tissues. During early embryonic development (E9.5-11.5), LacZ expression was detected in tissues where the endogenous Col2a1 transcript is expressed such as the otic capsule, notochord, developing brain, sclerotome and mesenchymal condensations of future cartilage. At later stages, Cre activity was observed in all cartilaginous tissues with virtually 100% of chondrocytes being LacZ positive. These data suggest that the Col2a1-Cre mouse strain described here can be useful to achieve Cre-mediated recombination in Col2a1 expressing cells, especially in chondrocytes.

Polymorphic insertions/deletions of both 1550nt and 100nt in two microsatellite-containing, LINE-related intronic regions of the rabbit kappa-casein gene.

The most frequent allele of the rabbit kappa-casein (kappa-Cas)-encoding gene (A allele) has previously been shown to possess two sequences similar to those found in the 5' end of long interspersed repeated elements (LINE). Part of an inverted rabbit LINE is present in the first intron and part of a direct rabbit LINE in the fourth intron. We describe herewith a less frequent allele (B allele) that lacks both 100bp in the first intron and 1550bp in the fourth intron. It was not possible to identify any allele exhibiting only one of the deletions in a population of 55 rabbits. The 100bp present in the first intron of the A allele, but absent from the B allele, are located at the 5' end of the inverse complementary LINE and include the poly (T) track of the LINE. The 1550bp present in the fourth intron of the A allele, but absent from the B allele, include the entire direct LINE sequence. Therefore, the B allele only possesses one partial LINE sequence that is located in the first intron and is truncated when compared to the copy found in the first intron of the A allele. The B allele might thus be more recent than the A allele. Differences between the sequences of transcripts corresponding to each allele are limited to two silent mutations and three modifications in the 3' UTR. In the mammary glands of lactating rabbits, which are homozygous for both alleles, kappa-Cas mRNA accumulate to similar levels and are translated into identical kappa-Cas that are secreted at similar concentrations into milk.

Structure of the rabbit kappa-casein encoding gene: expression of the cloned gene in the mammary gland of transgenic mice.

The rabbit kappa-casein (kappa-Cas) encoding gene has been isolated as a series of overlapping DNA fragments cloned from a rabbit genomic library constructed in bacteriophage lambda EMBL3. The clones harboured the 7.5-kb gene flanked by about 2.1 kb upstream and 9 kb downstream sequences. The cloned gene is the most frequently occurring of two kappa-Cas alleles identified in New Zealand rabbits. Comparison of the corresponding domains in rabbit and bovine kappa-Cas shows that both genes comprise 5 exons and that the exon/intron boundary positions are conserved whereas the introns have diverged considerably. The first three introns are shorter in the rabbit, the second intron showing the greatest difference between the two species: 1.35 kb instead of 5.8 kb in the bovine gene. Repetitive sequence motives reminiscent of the rabbit C type repeat and the complementary inverted C type repeat were identified in the fourth and first introns, respectively. Transgenic mice were produced by microinjecting into mouse oocytes an isolated genomic DNA fragment which contained the entire kappa-Cas coding region, together with 2.1-kb 5' and 4.0-kb 3' flanking region. Expression of transgene rabbit kappa-Cas mRNA could be detected in the mammary gland of lactating transgenic mice and the production of rabbit kappa-Cas was detected in milk using species-specific antibodies. The cloned gene is thus functional.

Characterization of rabbit kappa-casein cDNA: control of kappa-casein gene expression in vivo and in vitro.

The rabbit kappa-casein cDNA was cloned and sequenced. One of the isolated clones included almost the entire 5' end, while another clone corresponded to the 3' end of the cDNA. No polyadenylation site was found and therefore this clone did not harbour the complete cDNA. The amino acid sequence of a full-length protein was deduced from the nucleotide sequence obtained for this partial cDNA. It revealed the presence of a chymosin cleavage site and five potential phosphorylation sites. Rabbit kappa-casein was compared with those already described in other species. The rabbit sequence is closer to the ovine than to the mouse sequence. This result supports the idea that Lagomorpha are not closer to Rodentia than to Artiodactyla. The cDNA described above was used to study kappa-casein gene expression in the rabbit mammary gland. This expression was induced primarily by prolactin in mammary gland organoids and was similar to alpha s1-casein gene expression in vivo. The kappa-casein gene present in the casein gene locus is thus subject to the same regulation as the alpha s1-casein gene, although it has evolved from a fibrinogen gene.

[Detection of the kappa-casein genotype in bulls using restriction fragment length polymorphisms]. / Detekcia genotypu kapa-kazeínu u býkov hovädzieho dobytka na základe polymorfizmu dlzky restrikcných fragmentov (FFLP).

Genetic analysis of RFLP was used for detection of genotype and allele frequencies of kappa-casein in Slovakian Spotted (60 bulls) and Slovakian Pinzgau (22 bulls) cattle breeds, according to the method of Medrano et al. (1990). DNA was prepared from the semen of animals. In the Slovakian Spotted breed the frequencies of alleles were as follows: kappa-CnA = 0.666, kappa-CnB = 0.333. The frequencies of kappa-Cn A/A, A/B and B/B genotypes were 45.00, 43.33 and 11.66, respectively. In the 22 tested Pinzgau bulls, the frequencies of the A and B alleles were 0.682 and 0.318, respectively. The percentual occurrence of genotypes was also determined: 54.54 (kappa-Cn A/A), 27.27 (kappa-Cn A/B) and 18.18 (kappa-Cn B/B). Comparing our own results with those of Mácha et al. (1968), who carried out the analysis of distribution of the kappa-casein genetic variants in the same cattle breeds by starch gel electrophoresis of the milk samples of 170 cows (Tab. I), the 16 p.c. decrease of the allele B in the Slovakian Spotted cattle, lasting about 30 years, is very remarkable. The occurrence of homozygous genotype BB decreased by 35 p.c. In addition, the homozygous genotype AA increased by about 18 p.c. and the occurrence of heterozygous genotype is also higher by nearly 17 p.c. In the same comparison of the Slovakian Pinzgau breed, no difference was estimated in the allele frequencies of kappa-Cn (Tab. I).(ABSTRACT TRUNCATED AT 250 WORDS)

Generation of rabbit pluripotent stem cell lines.

Pluripotent stem cells have the capacity to divide indefinitely and to differentiate into all somatic cells and tissue lines. They can be genetically manipulated in vitro by knocking genes in or out, and therefore serve as an excellent tool for gene function studies and for the generation of models for some human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, many attempts have been made to generate pluripotent stem cell lines from other species. Comparative characterization of ESCs from different species would help us to understand differences and similarities in the signaling pathways involved in the maintenance of pluripotency and the initiation of differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved across different species. This report gives an overview of research into embryonic and induced pluripotent stem cells in the rabbit, an important nonrodent species with considerable merits as an animal model for specific diseases. A number of putative rabbit ESC and induced pluripotent stem cell lines have been described. All of them expressed stem cell-associated markers and maintained apparent pluripotency during multiple passages in vitro, but none have been convincingly proven to be fully pluripotent in vivo. Moreover, as in other domestic species, the markers currently used to characterize the putative rabbit ESCs are suboptimal because recent studies have revealed that they are not always specific to the pluripotent inner cell mass. Future validation of rabbit pluripotent stem cells would benefit greatly from a validated panel of molecular markers specific to pluripotent cells of the developing rabbit embryos. Using rabbit-specific pluripotency genes may improve the efficiency of somatic cell reprogramming for generating induced pluripotent stem cells and thereby overcome some of the challenges limiting the potential of this technology.

Expression of differentiated functions in dexamethasone-resistant hepatoma cells.

The expression of liver-specific functions of different dexamethasone-resistant variants derived from a well-differentiated dexamethasone-sensitive Reuber H35 rat hepatoma cell line (Faza 967) was examined during long-term cultivation. The dexamethasone-sensitive Faza 967 cells are characterized by the activity of tyrosine aminotransferase (TAT) and gluconeogenic enzymes, secretion of serum albumin, and the presence of liver isozymes of alcohol dehydrogenase (L-ADH), aldolase (aldolase-B), and five isoenzymes of lactate dehydrogenase (LDH). The hormone-resistant cells undergo a very dramatic change in expression of most liver-specific functions (dedifferentiation) during long-term culture, in contrast to the sensitive cells in which only certain functions (TAT activity, inducibility, and synthesis of serum albumin) exhibit considerable changes. The hormone-dependent growth sensitivity and the expression of other differentiated functions is not controlled in coordinated way in Faza 967 cells. The time course of the expression of liver-specific functions shows that the cells are resistant before they became 'dedifferentiated', i.e., loss of these liver-specific functions is not a prerequisite of the establishment of the hormone-resistant state.

Changes in the expression of differentiated functions during long-term cultivation of rat hepatoma cells.

The stability of the expression of six differentiated functions was examined during long-term cultivation of rat hepatoma cells. Faza 967 cell line--a clonal descendant of the Reuber H35 hepatoma--is characterized by the activity of tyrosine aminotransferase (TAT) and gluconeogenetic enzymes; secretion of serum albumin; and the presence of liver isozymes of alcohol dehydrogenase (ADH-L), aldolase (aldolase-B) and five isozymes of lactate dehydrogenase (LDH). During the 3-year-long cultivation of Faza 967 cells TAT specific activity, inducibility, and albumin production were reduced drastically whereas the expression of the three liver-specific isozymes examined was maintained. The majority of Faza 967 cells were able to perform gluconeogenesis after 3 years of continuous cultivation. Our results show that long-term cultivation of hepatoma cells may change the expression of certain liver-specific functions independently of the expression of other differentiated functions.