Generation of transgene-free mouse induced pluripotent stem cells using an excisable lentiviral system.

One goal of research using induced pluripotent stem cell (iPSC) is to generate patient-specific cells which can be used to obtain multiple types of differentiated cells as disease models. Minimally or non-integrating methods to deliver the reprogramming genes are considered to be the best but they may be inefficient. Lentiviral delivery is currently among the most efficient methods but it integrates transgenes into the genome, which may affect the behavior of the iPSC if integration occurs into an important locus. Here we designed a polycistronic lentiviral construct containing four pluripotency genes with an EGFP selection marker. The cassette was excisable with the Cre-loxP system making possible the removal of the integrated transgenes from the genome. Mouse embryonic fibroblasts were reprogrammed using this viral system, rapidly resulting in large number of iPSC colonies. Based on the lowest EGFP expression level, one parental line was chosen for excision. Introduction of the Cre recombinase resulted in transgene-free iPSC subclones. The effect of the transgenes was assessed by comparing the parental iPSC with two of its transgene-free subclones. Both excised and non-excised iPSCs expressed standard pluripotency markers. The subclones obtained after Cre recombination were capable of differentiation in vitro, in contrast to the parental, non-excised cells and formed germ-line competent chimeras in vivo.

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.

Nucleolar re-activation is delayed in mouse embryos cloned from two different cell lines.

Aim of this study was to evaluate and compare embryonic genome activation (EGA) in mouse embryos of different origin using nucleolus as a marker. Early and late 2-cell and late 4-cell stage embryos, prepared by in vitro fertilization (IVF), parthenogenetic activation (PG), and nuclear transfer of mouse embryonic fibroblast (MEF) and mouse HM1 embryonic stem cells (HM1), were processed for autoradiography following (3)H-uridine incubation (transcriptional activity), transmission electron microscopy (ultrastructure) and immunofluorescence (nucleolar proteins; upstream binding factor, UBF and nucleophosmin, B23). All early 2-cell embryos showed transcriptional activity only in nucleoplasm, not over nucleolar precursor bodies (NPBs). UBF was diffusely localized to cytoplasm and B23 to cytoplasm and nucleoplasm. Late 2-cell IVF and PG embryos displayed transcription over nucleoplasm and NPBs. Ultrastructurally, the latter were developing into functional nucleoli. NT-MEF and NT-HM1 embryos displayed transcription over nucleoplasm, but not over NPBs. Development of NPBs into nucleoli was lacking. UBF was in both groups localized to nucleoplasm or distinctly to presumptive NPBs. B23 was distinctly localized to NPBs. All 4-cell embryos presented nucleoplasmic transcription and developing fibrillo-granular nucleoli. UBF and B23 were distinctly localized to nucleoli. However, whereas fully transformed reticulated fibrillo-granular nucleoli were found in IVF and PG embryos, NT-MEF and -HM1 embryos displayed early NPBs transformation. In conclusion, despite normal onset of EGA in cloned embryos, activation of functional nucleoli was one cell cycle delayed in NT embryos. NT-MEF embryos displayed normal targeting but delayed activation of nucleolar proteins. Contrary, in NT-HM1 embryos, both of these processes were delayed.

Analysis of the applicability of molecular markers linked to the PVY extreme resistance gene Rysto, and the identification of new markers.

In this study molecular markers linked to the Rysto gene, which originates from the wild potato species Solanum stoloniferum and confers extreme resistance against PVY, were identified and the applicability of recently published Rysto, markers was analyzed. Three RAPD markers covering a total distance of 8.60 cM were detected in this experiment. The closest of these markers was located 0.53 cM from the gene. From among the published markers only one had diagnostic value in the experimental plant material, and mapped 2.95 cM from the gene, on the side opposite the RAPD markers developed in the present study. All the markers analyzed were present in Solanum stoloniferum accessions, irrespective of their resistance, indicating that these sequences are linked to the locus and not exclusively to the dominant allele of the Rysto gene in the wild species. The inapplicability of several published markers indicates that the genetic background is decisive in this tetraploid and highly heterozygous species. This means that it may be necessary to develop markers from the breeding material itself, until the resistance gene is not cloned and cannot be used as a selection marker in marker-assisted selection.

Possibility of the use of Solanum brevides based soft rot resistance in potato breeding.

In our experiments we dealt with the bacterial soft rot of potato caused by Erwinia species. In the experiments back cross progenies (BC1, BC2, BC3 and BC4) of Solanum brevidens + Solanum tuberosum somatic hybrids produced by the Potato Research Centre, Keszthely were tested to the infection of E. carotovora ssp. carotovora (Eca) and E. chrysanthemi (Echr). All together 29 BC genotypes pre selected from several hundred breeding lines based on their preferred agronomical appearance and virus resistance characters as well as 2 varieties (White lady and Hópehely) as controls were involved into the experiments. Tuber slices from each genotype were artificially infected after 2 and 5 months of harvest with bacteria suspension (Eca strain D3, and Echr strain CHR 1492) and incubated at 27 degrees C with 100% relative air humidity for 48 h before evaluation. Dry matter and starch content of tubers were determined right before the tests. Volume of rotted tuber tissue was determined in mm3 and used for comparison of the level of resistance or susceptibility of the genotypes. Relationship between the reaction to the bacteria strains and dry matter/starch content was examined also.

Tissue-specific signal(s) activate the promoter of a metallocarboxypeptidase inhibitor gene family in potato tuber and berry.

The molecular basis of the differential expression of the GM7-type metallocarboxypeptidase inhibitor (MCPI) genes in tuberizing (StMCPI) and non-tuberizing Solanum species (SbMCPI) was investigated. It was shown that the StMCPI is encoded by a gene family in Solanum tuberosum (potato), but SbMCPI might be a single-copy gene in the non-tuberizing species Solanum brevidens. The StMCPI promoter shows evolutionary relatedness to the S. brevidens-derived SbMCPI and to the fruit-specific tomato promoter 2A11. Both StMCPI and SbMCPI promoter regions were able to confer tuber- and berry-specific expression for the beta-glucuronidase reporter gene in potato suggesting that the difference in MCPI gene expression is in trans regulatory factors between the tuberizing and the non-tuberizing Solanum species. The MCPI promoters did not respond to metabolic, environmental or hormonal signals in leaves. Thus, the MCPI genes are regulated in a different way than the other known tuber-specific genes and potentially are suitable for biotechnological application in potato to provide specific transgene expression in tuber and berry.

Vigorous growth of fusion products allows highly efficient selection of interspecific potato somatic hybrids: molecular proofs.

An early identification of fusion products was based on the presumed vigorous growth of hybrid calluses after fusion between Solanum brevidens and S. tuberosum leaf protoplasts. The S. brevidens protoplasts were unable to form multicellular colonies under the applied culture conditions. Three size groups of calluses were separated and analyzed at two different early phases of culture period. "Squash blot" hybridization with a S. brevidens specific repetitive DNA probe showed that the group of the largest calluses consisted of putative somatic hybrids with a frequency of 80-100% in three independent experiments. Furthermore, approximately 80-95% of the middle sized calluses and 33-90% of the smallest ones were shown to be hybrid. The unexpectedly high percentage of fusion products, even in the case of the smallest calluses, may result from the suppression of the development of parental potato colonies in cultures with mixed cell population. Till this time 120 independent colonies selected as putative hybrids have been regenerated into plants. All of them exhibited hybrid phenotype, and their hybrid origin was proved by cytological and restriction fragment length polymorphism analyses.

Plant regeneration from callus and protoplast cultures of Helianthus giganteus L.

Methods of plant regeneration from callus and protoplasts of Helianthus giganteus L. are described. Embryogenic callus was obtained from leaf explants and plants were regenerated from these calli on MS media with different combinations of benzyladenine and naphtaleneacetic acid. Leaf protoplasts isolated from in vitro grown plants formed somatic embryos when cultured in agarose solidified droplets of V-KM medium containing benzyladenine and naphtaleneacetic acid. Embryos developed into plantlets on media with reduced auxin contents. Regenerated plants were successfully planted in soil.