Identification of survivin, an inhibitor of apoptosis, in canine urinary bladder transitional cell carcinoma.

Survivin, an inhibitor of apoptosis, is overexpressed in human invasive transitional cell carcinoma (TCC) of the urinary bladder. Survivin expression in canine TCC has not been defined. This study was designed to compare survivin expression between canine TCC and normal urinary bladder tissue. Reverse-transcriptase polymerase chain reaction (PCR) and immunohistochemistry (IHC) were performed on fresh-frozen and formalin-fixed tissues, respectively. All TCC tissues (n = 6) and 11/22 normal tissues assessed by PCR were positive for survivin. This difference was not significant (P = 0.06). With regard to IHC, 28/41 TCC samples were positive for nuclear survivin, whereas 0/46 normal tissues had nuclear immunoreactivity (P < 0.001). Cytoplasmic immunoreactivity did not significantly differ between TCC (7/41) and normal tissues (17/46) (P = 0.07). We conclude that nuclear survivin is present in canine TCC, but not in normal bladder urothelium. Future studies will evaluate the role of nuclear survivin in TCC development and as a potential therapeutic target.

Production of endothelial nitric oxide synthase (eNOS) over-expressing piglets.

Vascular function, vascular structure, and homeostasis are thought to be regulated in part by nitric oxide (NO) released by endothelial cell nitric oxide synthase (eNOS), and NO released by eNOS plays an important role in modulating metabolism of skeletal and cardiac muscle in health and disease. The pig is an optimal model for human diseases because of the large number of important similarities between the genomic, metabolic and cardiovascular systems of pigs and humans. To gain a better understanding of cardiovascular regulation by eNOS we produced pigs carrying an endogenous eNOS gene driven by a Tie-2 promoter and tagged with a V5 His tag. Nuclear transfer was conducted to create these animals and the effects of two different oocyte activation treatments and two different culture systems were examined. Donor cells were electrically fused to the recipient oocytes. Electrical fusion/activation (1 mM calcium in mannitol: Treatment 1) and electrical fusion (0.1 mM calcium in mannitol)/chemical activation (200 microM Thimerosal for 10 min followed by 8 mM DTT for 30 min: Treatment 2) were used. Embryos were surgically transferred to the oviducts of gilts that exhibited estrus on the day of fusion or the day of transfer. Two cloned transgenic piglets were born from Treatment 1 and low oxygen, and another two from Treatment 2 and normal oxygen. PCR, RT-PCR, Western blotting and immunohistochemistry confirmed that the pigs were transgenic, made message, made the fusion protein and that the fusion protein localized to the endothelial cells of placental vasculature from the conceptuses as did the endogenous eNOS. Thus both activation conditions and culture systems are compatible with development to term. These pigs will serve as the founders for a colony of miniature pigs that will help to elucidate the function of eNOS in regulating muscle metabolism and the cardiorespiratory system.

Effect of epidermal growth factor and insulin-like growth factor I on porcine preantral follicular growth, antrum formation, and stimulation of granulosal cell proliferation and suppression of apoptosis in vitro.

The object of this study was to investigate the role of epidermal growth factor (EGF) and IGF-I in the regulation of preantral follicular growth, antrum formation, and granulosal cell proliferation/ apoptosis. Porcine preantral follicles were manually dissected and cultured for up to 8 d in Waymouth's (Exp. 1) or alpha-minimum Eagle's essential medium (Exp. 2 and 3) supplemented with 10 microg/mL of transferrin, 100 microg/mL of L-ascorbic acid, and 2 mU/mL of ovine FSH, in the presence (Exp. 1 and 3) or absence (Exp. 2) of 7.5% fetal calf serum. According to the experimental protocol, IGF-I (0, 1, 10, or 100 ng/mL; Exp. 1), or IGF-I (50 ng/mL), EGF (10 ng/mL) and EGF+IGF-I (Exp. 2 and 3) were added to the culture media. In Exp. 1, follicles exhibited a concentration-dependent response (P < 0.05) to IGF-I, with the highest rates of granulosal cell proliferation, follicular integrity, and recovery rate of cumulus cell-oocyte complexes and lowest incidence of apoptosis occurring at the highest IGF-I dose. In Exp. 2 serum-free medium, granulosal cell proliferation was low (1 to 5%), irrespective of whether EGF and/or IGF-I were present and cellular apoptosis was increased (P < 0.05) on d 4 and 8 in the EGF+IGF-I group compared with the addition of either factor alone. In Exp. 3, granulosal cell proliferation was high in all follicles cultured in serum-containing medium for the first 3 d, but fell sharply (P < 0.05) on d 4, except in media containing IGF-I. Collectively, EGF and IGF-I increased granulosal cell proliferation, decreased apoptosis, and promoted follicular antrum formation. These results may provide useful information for developing a preantral follicular culture system in which the oocytes are capable of fertilization and embryonic development.

Conditional deletion of the bcl-x gene from mouse mammary epithelium results in accelerated apoptosis during involution but does not compromise cell function during lactation.

In the mammary gland Bcl-x is the most abundant cell survival factor from the Bcl-2 family. Since Bcl-x null mice die around day 12 of embryogenesis, the relevance of this protein in organ development and function is poorly understood. In erythroid cells bcl-x gene expression is controlled by cytokines and the transcription factor Stat5 (signal transducer and activator of transcription). However, we identified that bcl-x RNA levels in mammary tissue from prolactin receptor- and Stat5-null mice were indistinguishable from wild type mice. We have proposed that Bcl-x might control the survival of mammary epithelial cells throughout pregnancy, lactation, and the early stages of involution, and we have now tested this hypothesis through the conditional deletion of the bcl-x gene from mouse mammary epithelium. Conditional (floxed) bcl-x alleles were excised from alveolar cells during pregnancy using a Cre transgene under the control of the whey acidic protein gene promoter. Deletion of the bcl-x gene from the entire epithelial compartment (ducts and alveoli) was achieved by expressing Cre-recombinase under control of the mouse mammary tumor virus long terminal repeat. The absence of Bcl-x did not compromise proliferation and differentiation of mammary ductal and alveolar epithelial cells in virgin mice and during pregnancy and lactation. However, epithelial cell death and tissue remodeling were accelerated in the bcl-x conditional knockout mice during the first stage of involution. Concomitant deletion of the bax gene did not significantly modify the Bcl-x phenotype. Our results suggest that Bcl-x is not essential during mammopoiesis, but is critical for controlled apoptosis during the first phase of involution.

Conditional deletion of the Bcl-x gene from erythroid cells results in hemolytic anemia and profound splenomegaly.

Bcl-x is a member of the Bcl2 family and has been suggested to be important for the survival and maturation of various cell types including the erythroid lineage. To define the consequences of Bcl-x loss in erythroid cells and other adult tissues, we have generated mice conditionally deficient in the Bcl-x gene using the Cre-loxP recombination system. The temporal and spatial excision of the floxed Bcl-x locus was achieved by expressing the Cre recombinase gene under control of the MMTV-LTR. By the age of five weeks, Bcl-x conditional mutant mice exhibited hyperproliferation of megakaryocytes and a decline in the number of circulating platelets. Three-month-old animals suffered from severe hemolytic anemia, hyperplasia of immature erythroid cells and profound enlargement of the spleen. We demonstrate that Bcl-x is only required for the survival of erythroid cells at the end of maturation, which includes enucleated reticulocytes in circulation. The extensive proliferation of immature erythroid cells in the spleen and bone marrow might be the result of a fast turnover of late red blood cell precursors and accelerated erythropoiesis in response to tissue hypoxia. The increase in cell death of late erythroid cells is independent from the proapoptotic factor Bax, as demonstrated in conditional double mutant mice for Bcl-x and Bax. Mice conditionally deficient in Bcl-x permitted us for the first time to study the effects of Bcl-x deficiency on cell proliferation, maturation and survival under physiological conditions in an adult animal.

Bcl-x and Bax regulate mouse primordial germ cell survival and apoptosis during embryogenesis.

Restricted germ cell loss through apoptosis is initiated in the fetal gonad around embryonic day 13.5 (E13.5) as part of normal germ cell development. The mechanism of this germ cell attrition is unknown. We show that Bcl-x plays a crucial role in maintaining the survival of mouse germ cells during gonadogenesis. A bcl-x hypomorphic mouse was generated through the introduction of a neomycin (neo) gene into the promoter of the bcl-x gene by homologous recombination. Mice that contained two copies of the hypomorphic allele had severe reproductive defects attributed to compromised germ cell development. Males with two mutant alleles lacked spermatogonia and were sterile; females showed a severely reduced population of primordial and primary follicles and exhibited greatly impaired fertility. Primordial germ cells (PGCs) in bcl-x hypomorph mice migrated to the genital ridge by E12.5 but were depleted by E15.5, a time when Bcl-x and Bax were present. Two additional bcl-x transcripts were identified in fetal germ cells more than 300 bp upstream of previously reported start sites. Insertion of a neo cassette led to a down-regulation of the bcl-x gene at E12.5 in the hypomorph. Bax was detected by immunohistochemistry in germ cells from bcl-x hypomorph and control testes at E12.5 and E13.5. Bcl-x function was restored, and animals of both genders were fertile after removal of the neo selection cassette using Cre-mediated recombination. Alternatively, the loss of Bcl-x function in the hypomorph was corrected by the deletion of both copies of the bax gene, resulting in a restoration of germ cell survival. These findings demonstrate that the balance of Bcl-x and Bax control PGC survival and apoptosis.

Genomic architecture and transcriptional activation of the mouse and human tumor susceptibility gene TSG101: common types of shorter transcripts are true alternative splice variants.

The functional inactivation of the tumor susceptibility gene tsg101 in mouse NIH3T3 cells leads to cell transformation and the formation of metastatic tumors in nude mice. We cloned, mapped and sequenced the mouse tsg101 gene and further identified a processed pseudogene that is 98% identical to the tsg101 cDNA. Based on Northern blot analysis, tsg101 is expressed ubiquitously in mouse tissues. A comparison of the coding region of the mouse tsg101 gene with the human TSG101 cDNA revealed that both the mouse and human gene encode ten additional highly conserved amino acids at the N-terminus. Based on the mouse tsg101 genomic structure, we predicted four additional introns within the human TSG101 gene. Their location was confirmed using PCR and sequencing analysis. The presence of these so far unidentified introns now explains published data on aberrantly spliced mRNA products that were frequently observed in primary breast tumors. We show that a majority of shorter TSG101 transcripts are not the result of aberrant splicing events, but represent a fraction of true alternative splice variants. Finally, we examined tsg101 expression patterns during different stages of mammary gland development and in different transgenic mouse models for breast tumorigenesis.

Isolation and genetic characterization of the porcine apolipoprotein E gene.

The present report describes the isolation and genetic characterization of the porcine apolipoprotein E (apo-E) gene. A single positive recombinant phage clone containing a 10.7-kb insert was isolated from a porcine genomic library, and a 4.2-kb fragment was subcloned and sequenced. The 4.2-kb fragment contained the entire apo-E gene in addition to upstream and downstream sequences (GenBank accession no. 470240). The porcine apo-E gene is made up of four exons and three introns, and encodes a preapo-E protein comprised of a signal peptide of 18 amino acids and a mature protein of 299 amino acids. The porcine apo-E gene contains a (CG)13 microsatellite marker within intron three. This microsatellite is moderately polymorphic, and at least four alleles were evident at this locus among 10 animals from each of the Yorkshire, Hampshire, Landrace and Duroc breeds. Finally, localization of the porcine apo-E gene to chromosome 6 band q2.1 was determined by fluorescent in situ hybridization and confirmed by genetic linkage analysis.

Factors affecting the efficiency of introducing precise genetic changes in ES cells by homologous recombination: tag-and-exchange versus the Cre-loxp system.

The introduction of genetic modifications in specific genes by homologous recombination provides a powerful tool for elucidation of structure-function relationships of proteins of biological interest. Presently, there are several alternative methods of homologous recombination that permit the introduction of small genetic modifications in specific loci. Two of the most widely used methods are the tag-and-exchange, based on the use of positive-negative selection markers, and the Cre-loxP system, based on the use of a site-specific recombinase. The efficiency of detection of targeting events at different loci using the two systems was compared. Additionally, we analysed how the distance between two gene markers placed within the region of homology of a targeting vector affects the rate at which both markers are introduced into the locus during the homologous recombination event. Our results indicate that the method based on the use of positive-negative selection markers was less efficient than the Cre-loxP based system, irrespective of locus or type of positive-negative selection. It was also determined that as the distance between the selectable marker and the genetic modification being introduced increases, there is a progressive reduction in the efficiency of detecting events with the desired genetic modification.

Cre-mediated recombination at the murine whey acidic protein (mWAP) locus.

The mouse whey acidic protein (WAP) gene in mouse embryonic stem (ES) cells has been targeted with a loxP-flanked neomycin phosphotransferase-thymidine kinase (neo-TK) cassette inserted into exon 4. Southern blot revealed that 51 of 199 colonies were correctly targeted (1:4). Next, a Cre-encoding plasmid was electroporated into a targeted cell line to cause the deletion of the neo-TK cassette. Modified ES cell colonies were identified by polymerase chain reaction (PCR); 44 out of 50 colonies (88%) had undergone Cre-mediated deletion. Finally, a loxP-tagged cell line was co-electroporated with a Cre-encoding plasmid and a loxP-containing neo plasmid for site-specific insertion into the WAP locus. The frequency of this event was 23% (11 of 48) of that obtained with random integration. This demonstrates the feasibility of using the Cre-loxP system for site-specific integration in ES cells. Moreover, this is the first report of targeting a loxP-containing transgene into a predetermined location in ES cells. Ultimately, a mouse model derived from these modified ES cells will usher in a second generation of animal "bioreactor" models where the inserted transgene is controlled exclusively by the endogenous locus regulatory elements. In addition, oncogenesis can be explored from single copy oncogene/tumor suppressor gene inserts, which are regulated in a temporal and tissue-specific manner. It is hoped that regulation of transgene expression in this fashion will help elucidate the underlying mechanisms of normal development in the mammary gland.

Truncated fibronectin. An autologous growth-promoting substance secreted by renal carcinoma cells.

The human renal carcinoma cell (RCC) line ACHN proliferates in the absence of exogenous growth factors and secretes a 178-kilodalton growth-promoting substance (GPS). Complementary DNA (cDNA) was isolated that coded for polypeptides antigenically cross-reactive with GPS. Nucleotide sequencing of the cDNA showed strong homology with human fibronectin (FN). The deletion of an adenine in human FN codon 1482 caused a reading-frame shift that predicted early termination of translation after 1518 amino acid residues. Western immunoblotting human FN and GPS with anti-human FN antibodies showed that GPS was a truncated FN. Previous work found that malignant cells synthesized, bound, and deposited into the extracellular matrix decreased amounts of FN. Addition of this substance to transformed cells changed their morphology but not their rate of growth. By contrast, partial proteolysis of FN resulted in a prominent 180-kilodalton fragment that stimulated DNA synthesis. Similar to this finding, the authors showed that truncation of fibronectin during synthesis appeared to unmask latent DNA synthetic stimulating activity. Polymerase chain reaction methods using genomic DNA from normal kidneys and RCC and primers specific for the GPS-human FN gene showed two products of identical size, indicating that genomic amplification did not cause activation of the human FN gene in RCC to produce GPS. Restriction-fragment length analysis demonstrated identical patterns in DNA extracted from both normal kidneys and RCC, suggesting that chromosomal rearrangements did not activate the GPS-human FN gene. This study showed that genetic changes detectable only by DNA sequencing can explain the activation of the normal human FN gene to produce GPS, a product important for autologous growth stimulation of RCC.

The nucleotide sequence of the luxA and luxB genes of Xenorhabdus luminescens HM and a comparison of the amino acid sequences of luciferases from four species of bioluminescent bacteria.

The luxA and luxB genes of bioluminescent bacteria encode the alpha and beta subunits of luciferase, respectively. Sequences of the luxA and luxB genes of Xenorhabdus luminescens, the only terrestrial bioluminescent bacterium known, were determined and the amino acid sequence of luciferase deduced. The alpha subunit was found to contain 360 amino acids and has a calculated molecular weight of 41,005 Da, while the beta subunit contains 327 amino acids and has a calculated molecular weight of 37,684 Da. Alignment of this luciferase with the luciferases of three marine bacteria showed 196 (or 55%) conserved residues in the alpha subunit and 114 (or 35%) conserved residues in the beta subunit. The highest degree of homology between any two species was between the luciferases of X. luminescens and Vibrio harveyi with 84% identity in the alpha subunits and 59% identity in the beta subunits.