Gregory H. Leno

Adeno-associated virus-targeted disruption of the CFTR gene in cloned ferrets.

Somatic cell gene targeting combined with nuclear transfer cloning presents tremendous potential for the creation of new, large-animal models of human diseases. Mouse disease models often fail to reproduce human phenotypes, underscoring the need for the generation and study of alternative disease models. Mice deficient for CFTR have been poor models for cystic fibrosis (CF), lacking many aspects of human CF lung disease. In this study, we describe the production of a CFTR gene-deficient model in the domestic ferret using recombinant adeno-associated virus-mediated gene targeting in fibroblasts, followed by nuclear transfer cloning. As part of this approach, we developed a somatic cell rejuvenation protocol using serial nuclear transfer to produce live CFTR-deficient clones from senescent gene-targeted fibroblasts. We transferred 472 reconstructed embryos into 11 recipient jills and obtained 8 healthy male ferret clones heterozygous for a disruption in exon 10 of the CFTR gene. To our knowledge, this study represents the first description of genetically engineered ferrets and describes an approach that may be of substantial utility in modeling not only CF, but also other genetic diseases.

Identification of Differentially Expressed Genes in Individual Bovine Preimplantation Embryos Produced by Nuclear Transfer: Improper Reprogramming of Genes Required for Development

Abstract Using an interwoven-loop experimental design in conjunction with highly conservative linear mixed model methodology using estimated variance components, 18 genes differentially expressed between nuclear transfer (NT)- and in vitro fertilization (IVF)-produced embryos were identified. The set is comprised of three intermediate-filament protein genes (cytokeratin 8, cytokeratin 19, and vimentin), three metabolic genes (phosphoribosyl pyrophosphate synthetase 1, mitochondrial acetoacetyl-coenzyme A thiolase, and α-glucosidase), two lysosomal-related genes (prosaposin and lysosomal-associated membrane protein 2), and a gene associated with stress responses (heat shock protein 27) along with major histocompatibility complex class I, nidogen 2, a putative transport protein, heterogeneous nuclear ribonuclear protein K, mitochondrial 16S rRNA, and ES1 (a zebrafish orthologue of unknown function). The three remaining genes are novel. To our knowledge, this is the first report comparing individual embryos produced by NT and IVF using cDNA microarray technology for any species, and it uses a rigorous experimental design that emphasizes statistical significance to identify differentially expressed genes between NT and IVF embryos in cattle.

Efficient Term Development of Vitrified Ferret Embryos Using a Novel Pipette Chamber Technique

Abstract Development of an efficient cryopreservation technique for the domestic ferret is key for the long-term maintenance of valuable genetic specimens of this species and for the conservation of related endangered species. Unfortunately, current cryopreservation procedures, such as slow-rate freezing and vitrification with open pulled straws, are inefficient. In this report, we describe a pipette tip-based vitrification method that significantly improves the development of thawed ferret embryos following embryo transfer (ET). Ferret embryos at the morula (MR), compact morula (CM), and early blastocyst (EB) stages were vitrified using an Eppendorf microloader pipette tip as the chamber vessel. The rate of in vitro development was significantly (P < 0.05) higher among embryos vitrified at the CM (93.6%) and EB (100%) stages relative to those vitrified at the MR stages (58.7%). No significant developmental differences were observed when comparing CM and EB vitrified embryos with nonvitrified control CM (100%) and EB (100%) embryos. In addition, few differences in the ultrastructure of intracellular lipid droplets or in microfilament structure were observed between control embryos and embryos vitrified at any developmental stage. Vitrified-thawed CM/EB embryos cultured for 2 or 16 h before ET resulted in live birth rates of 71.3% and 77.4%, respectively. These rates were not significantly different from the control live birth rate (79.2%). However, culture for 32 h (25%) or 48 h (7.8%) after vitrification significantly reduced the rate of live births. These data indicate that the pipette chamber vitrification technique significantly improves the live birth rate of transferred ferret embryos relative to current state-of-the-art methods..

Chromatin Configurations in the Ferret Germinal Vesicle that Reflect Developmental Competence for In Vitro Maturation

In several mammalian species, the configuration of germinal vesicle (GV) chromatin correlates with the developmental competence of oocytes. Yet, no study has been published on the configuration of GV chromatin in ferret, nor is it known whether a specific configuration predicts meiotic competence in this species, in spite of the potential importance of ferret cloning to the study of human disease and to species conservation efforts. Here, we report on an analysis of the chromatin configuration in ferret GV oocytes and on how they correlate with meiotic development. Three distinct configurations were identified based on the degree of chromatin condensation: (1) fibrillar chromatin (FC), featuring strands of intertwined chromatin occupying most of the visible GV region; (2) intermediate condensed chromatin (ICC), characterized by dense, irregular chromatin masses throughout the GV; and (3) condensed chromatin (CC), which is highly compact and centered around the nucleolus. We also found that chromatin configuration was related to the extent of association with cumulus cells in cumulus-oocyte complexes; CC-configured oocytes were most often surrounded by a compact cumulus layer and also a compact corona but FC-configured oocytes were associated with neither. In addition, increasing chromatin condensation corresponded to an increase in oocyte diameter. Finally, following in vitro culture, significantly more CC-configured oocytes underwent maturation to meiotic metaphase II than did FC- or ICC-configured oocytes. We conclude that, in ferret, chromatin condensation is related to the sequential achievement of meiotic competencies during oocyte growth and differentiation, and thus can be used as a predictor of competence.

Indexing TNF-α gene expression using a gene-targeted reporter cell line

BackgroundCurrent cell-based drug screening technologies utilize randomly integrated reporter genes to index transcriptional activity of an endogenous gene of interest. In this context, reporter expression is controlled by known genetic elements that may only partially capture gene regulation and by unknown features of chromatin specific to the integration site. As an alternative technology, we applied highly efficient gene-targeting with recombinant adeno-associated virus to precisely integrate a luciferase reporter gene into exon 1 of the HeLa cell tumor necrosis factor-alpha (TNF-α) gene. Drugs known to induce TNF-α expression were then used to compare the authenticity of gene-targeted and randomly integrated transcriptional reporters.ResultsTNF-α-targeted reporter activity reflected endogenous TNF-α mRNA expression, whereas randomly integrated TNF-α reporter lines gave variable expression in response to transcriptional and epigenetic regulators. 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), currently used in cancer clinical trials to induce TNF-α gene transcription, was only effective at inducing reporter expression from TNF-α gene-targeted cells.ConclusionWe conclude that gene-targeted reporter cell lines provide predictive indexing of gene transcription for drug discovery.