The success of somatic nuclear transfer critically depends on the cell cycle stage of the donor nucleus and the recipient cytoplast. In this study we tested serum deprivation as well as two reversible cell cycle inhibitors, aphidicolin and butyrolactone I, for their ability to synchronize porcine fetal fibroblasts at either G0 stage or G1/S or G2/M transition. The synchronization efficiency of the various protocols was determined by fluorescence-activated cell sorting (FACS), cell proliferation assays, and semiquantitative multiplex reverse transcription-polymerase chain reaction detection of the cell cycle-regulated porcine Polo-like kinase mRNA (Plk-p). FACS measurements revealed that 66.6-73.3% of the porcine fetal fibroblasts were in G0/G1 stage (2C DNA content) in serum-supplemented medium. Short periods of 24-72 h of serum deprivation significantly increased the proportion of cells at G0/G1 phase to 77.9-80.2%, and mitotic activity had already terminated after 48 h. Prolonged culture in serum-deprived medium induced massive DNA fragmentation. Aphidicolin treatment led to an accumulation of 81.9 +/- 4.9% of cells at the G1/S transition. Butyrolactone I arrested 81.0 +/- 5.8% of the cells at the end of G1 stage and 37.0 +/- 6.8% at the G2/M transition. The effects of both chemical inhibitors were fully reversible, and their removal led to a rapid progression in the cell cycle. The measurement of Plk-p expression allowed discrimination between the presumptive G0 phase induced by serum deprivation and the G1/S transition arrest achieved by chemical inhibitors. These data indicate that porcine fetal fibroblasts can be effectively synchronized at various cell cycle stages without compromising their proliferation capacity.

Persistent infection by hepatitis B virus (HBV) is epidemiologically correlated with the prevalence of hepatocellular carcinoma, but its role in tumor development is not yet understood. To study the putative oncogenic potential of HBV, a non‐malignant immortal mouse hepatocyte line FMH202 harboring metallothionein promoter‐driven simian virus 40 large tumor antigen was transfected with HBV DNA. All stably transfected clones which replicated HBV displayed malignant growth characteristics in soft agar and were tumorigenic upon inoculation in nude mice. The nude mice tumors were histologically classified as differentiated or anaplastic hepatocellular carcinomas. As with human liver carcinomas, rearrangements of in vitro integrated HBV sequences were observed in the nude mouse tumors, and in tumor‐derived cell lines. In one case, expression of viral core and surface antigens was blocked in the tumors, correlating with hypermethylation of the HBV genome. However, the expression of X gene was maintained in most tumors and tumor‐derived cell lines. X protein was detected in nuclei by immune fluorescence and by immune blot. These results provide the first demonstration that HBV displays oncogenic potential in an experimental system. This system could be useful to functionally identify HBV genes which convey a tumorigenic phenotype.

entla (ent) is a novel recessive phenotype of mice. The underlying mutation was mapped to chromosome 9 (60.1 centimorgans) and identified as an allele of the Cacna2d2 gene encoding the ␣2␦-2 subunit of voltagegated calcium channels. The Cacna2d2 entla allele harbors a 38-kb duplication comprising the 117 nucleotides of exon 3. The predicted duplication of 39 amino acid residues near the subunit's N terminus results in the expression of a full-length, membrane-associated protein. Western blot data were consistent with correct cleavage of the ␣2␦-2 entla precursor into ␣2 entla and ␦2 proteins but indicated loss of the disulfide linkage between the two proteins. ent/ent mice develop ataxia by postnatal day 13-15, followed by paroxysmal dyskinesia a few days later. Two distinct types of cortical and hippocampal epileptic activity at 2 and 4 Hz were recorded, indicative of absence epilepsy. Homozygotes display reduced size and weight, increased mortality before weaning, and female infertility. No overt neuroanatomical abnormalities were detected. Ca 2؉ current densities recorded from acutely dissociated Purkinje cells of homozygous entla animals were reduced by 50% compared with wild type. Ligand binding assays using the antiepileptic drug [ 3 H]gabapentin, a specific ligand of the ␣2␦-1 and ␣2␦-2 subunits, revealed a >60% reduced maximum binding to cerebellar membranes of ent/ent compared with unaffected littermates. entla is allelic to ducky and ducky 2J , representing the third murine Cacna2d2 allele identified and so far the only one encoding an untruncated protein that is incorporated into membranes.Voltage-gated calcium channels (VGCCs) 1 mediate entry of Ca 2ϩ into excitable cells, thereby initiating a multitude of cellular processes including the release of neurotransmitter into the synaptic cleft. Based on their biophysical properties, VGCCs are subdivided into L-, N-, P/Q-, R-, and T-type channels. VGCCs are protein complexes consisting of a channelforming, voltage-sensing ␣1 subunit and ␣2␦, ␤, and ␥ auxiliary subunits (1, 2). Mutations in VGCC genes underlie various human and murine neurological diseases. Familial hemiplegic migraine (OMIM 602481), spinocerebellar ataxia (OMIM 183086, 603516, and 604432), and idiopathic generalized epilepsy (OMIM 600669) result from mutations in the ␣1A and ␤4 subunits. In mice, mutations in ␣1, ␤, ␥, and ␣2␦ subunits have been described (3). The ␣1A mutants tottering (4) and rocker (5), the ␤4 mutant lethargic (6), the ␥2 mutant stargazer (7), and the ␣2␦-2 mutants ducky and ducky 2J (8) all suffer from ataxia, paroxysmal dyskinesia, and epileptic spike wave discharges.Each ␣2␦ subunit is encoded by a single gene and posttranslationally cleaved into a long, N-terminal, extracellular ␣2 protein and a shorter, membrane-anchored ␦ polypeptide; the ␣2 and ␦ proteins are covalently linked by disulfide bonds (9). Upon recombinant coexpression with ␣1, ␤, and ␥ subunits, ␣2␦ subunits modulate channel activity by increasing calcium current density, shifting the voltage dependen...

The critical shortage of human donor organs has generated growing interest for porcine to human xenotransplantation. The major immunological barrier to xenotransplantation is the hyperacute rejection (HAR) response that is mediated by preformed xenoreactive antibodies and complement. A promising strategy to control the complement activation, is the expression of human complement regulatory proteins in transgenic animals. We have used the human early cytomegalovirus (CMV) promoter to drive expression of the human complement regulatory protein CD59 (hCD59) in transgenic pigs. A total of eight live transgenic founder animals was born from which five transgenic lines could be established. mRNA analysis and Western blotting revealed high expression of hCD59 in heart, kidney, skeletal muscle, and skin in animals of lines 1 and 5, as well as in the pancreas of four lines. This pattern of expression was confirmed by immunhistological staining. A cell-specific expression in heart and kidney tissue of transgenic lines 1 and 5 was determined. Primary fibroblasts and endothelial cell cultures derived from the aorta of transgenic pigs showed a significantly diminished sensitivity against the challenge with xenoreactive human antibodies and complement whereas non-transgenic control cells were highly susceptible to complement mediated lysis. Ex vivo perfusion of kidneys with pooled human blood revealed a significant protective effect of hCD59 against HAR. The average survival of transgenic kidneys was significantly extended (P<0.05) over nontransgenic controls (207.5+/-54.6 vs. 57.5+/-64.5 min). These data support the concept that hCD59 protects nonprimate cells against human complement mediated lysis and suggest that donor pigs transgenic for hCD59 could play a crucial role in clinical xenotransplantation. Two of five hCD59 transgenic lines showed strong hCD59 expression in several organs relevant for xenotransplantation and a protective effect against HAR. This indicates that the use of the CMV-promoter can facilitate the selection process for optimized transgene expression.

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