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.

Transgenic rabbit production with simian immunodeficiency virus-derived lentiviral vector.

Transgenic rabbit is the preferred disease model of atherosclerosis, lipoprotein metabolism and cardiovascular diseases since upon introducing genetic mutations of human genes, rabbit models reflect human physiological and pathological states more accurately than mouse models. Beyond that, transgenic rabbits are also used as bioreactors to produce pharmaceutical proteins in their milk. Since in the laboratory rabbit the conventional transgenesis has worked with the same low efficiency in the last twenty five years and truly pluripotent embryonic stem cells are not available to perform targeted mutagenesis, our aim was to adapt lentiviral transgenesis to this species. A simian immunodeficiency virus based replication defective lentiviral vector was used to create transgenic rabbit through perivitelline space injection of fertilized oocytes. The enhanced green fluorescent protein (GFP) gene was placed under the ubiquitous CAG promoter. Transgenic founder rabbits showed mosaic pattern of GFP expression. Transgene integration and expression was revealed in tissues derived from all three primary germ layers. Transgene expression was detected in the developing sperm cells and could get through the germ line without epigenetic silencing, albeit with very low frequency. Our data show for the first time, that lentiviral transgenesis could be a feasible and viable alternative method to create genetically modified laboratory rabbit.

Astroglia-derived retinoic acid is a key factor in glia-induced neurogenesis.

Astroglial cells are essential components of the neurogenic niches within the central nervous system. Emerging evidence suggests that they are among the key regulators of postnatal neurogenesis. Although astrocytes have been demonstrated to possess the potential to instruct stem cells to adopt a neuronal fate, little is known about the nature of the glia-derived instructive signals. Here we propose that all-trans retinoic acid, one of the most powerful morphogenic molecules regulating neuronal cell fate commitment, may be one of the glia-derived factors directing astroglia-induced neurogenesis. According to data obtained from several complementary approaches, we show that cultured astrocytes express the key enzyme mRNAs of retinoic acid biosynthesis and actively produce all-trans retinoic acid. We show that blockage of retinoic acid signaling by the pan-RAR antagonist AGN193109 prevents glia-induced neuron formation by noncommitted stem cells. Therefore, we provide strong in vitro evidence for retinoic acid action in astroglia-induced neuronal differentiation.

Effect of swim up and Percoll treatment on viability and acrosome integrity of frozen-thawed bull spermatozoa.

The aim of this study was to investigate the effect of the swim up and Percoll methods to select frozen-thawed bull spermatozoa with high quality membrane and acrosomal integrity and final concentration. Semen samples from six Holstein-Friesian bulls were examined. The whole experiment was repeated three times. Before and after both treatments, spermatozoa were subjected to a double-staining method and evaluated by brightfield light microscope using 40x dry, or 100x oil immersion objectives. The concentration of spermatozoa evaluated by haemocytometer was 8.8 x 10(7)/ml after thawing, and the percentage of live cells with intact acrosome was 45.8%. Both treatments significantly increased the proportion of live spermatozoa compared with no treatment, and the use of Percoll gradient resulted in a significantly higher percentage of living cells with an intact acrosome (88.2%) than the swim up method (69.4%). The concentration of spermatozoa after Percoll separation (9.3 x 10(6)/ml) was higher than that after the swim up method (5.8 x 10(6)/ml). These results indicate that spermatozoa with a higher viability and acrosome integrity can be obtained by Percoll separation than by the swim up method. Therefore the use of Percoll-treated spermatozoa in IVF systems can be more expedient.

Simultaneous evaluation of viability and acrosome integrity of mouse spermatozoa using light microscopy.

Determination of the percentage of live cells with intact acrosomes and no morphologic aberrations could be a practical index of semen quality. We applied viability and acrosome staining techniques, originally described for bull, boar and rabbit sperm, to mouse spermatozoa. The viability stain was either trypan blue or Congo red. The stain was precipitated by neutral red in the fixative. The acrosome was stained by Giemsa. Sperm morphology, including cytoplasmic droplets, could be evaluated as well. The staining method described here is a useful routine tool for simultaneous evaluation of the plasma membrane integrity of different sperm subdomains, the status of the acrosome, and cellular morphology.

Preimplantation genetic diagnosis in cattle: a review.

Preimplantation Genetic Diagnosis (PGD) is reviewed and novel fields where it may be applied are investigated. Technical advances of PGD in cattle embryos have already enabled its integration as a part of the MOET (Multiple Ovulation Embryo Transfer) breeding system. PGD for well-defined selection targets can enhance cattle breeding and embryo trade. It allows embryo selection according to their sex, and it may be used to breed special cow lines, or top bulls, by selecting embryos for valuable production traits using Marker Assisted Selection (MAS). A good allelic profile and/or the insertion of a transgene can be detected by PGD. This review article presents the technical requirements for PGD, and shows that this biotechnological method has great economic potential.

Embryonic stem cells alone are able to support fetal development in the mouse.

The developmental potential of embryonic stem (ES) cells versus 3.5 day inner cell mass (ICM) was compared after aggregation with normal diploid embryos and with developmentally compromised tetraploid embryos. ES cells were capable of colonizing somatic tissues in diploid aggregation chimeras but less efficiently than ICMs of the same genotype. When ICM in equilibrium with tetraploid and ES in equilibrium with tetraploid chimeras were made, the newborns were almost all completely ICM- or ES-derived, as judged by GPI isozyme analysis, but tetraploid cells were found in the yolk sac endoderm and trophectoderm lineage. Investigation of ES contribution in 13.5 day ES in equilibrium with tetraploid chimeras by DNA in situ hybridization confirmed the complete tetraploid origin of the placenta (except the fetal blood and blood vessels) and the yolk sac endoderm. However, the yolk sac mesoderm, amnion and fetus contained only ES-derived cells. ES-derived newborns failed to survive after birth, although they had normal birthweight and anatomically they appeared normal. This phenomenon remains unexplained at the moment. The present results prove that ES cells are able to support complete fetal development, resulting in ES-derived newborns, and suggest a useful route for studying the development of genetically manipulated ES cells in all fetal lineages.