How farm animals are improving human health and welfare.

The contribution of farm animals to human health and welfare cannot be properly addressed without reflecting on the impact that animal domestication has had upon human civilisation. About 14,000 years ago, the Neolithic revolution started with the domestication of animals and plants, resulting in the emergence of the main agricultural breeds of livestock and crops. In contrast, the breeding of new animal species for biomedical research, such as small rodents and other model species, is a relatively recent activity. The cellular and molecular mechanisms of inheritance have only been understood over the past few decades and translated into approaches to improve breeding success. In recent years, seminal discoveries in the fields of cellular reprogramming, genetic engineering, and whole-genome sequencing have accelerated this development. The first therapeutic proteins produced by biopharming in livestock have been approved to treat human patients. The suitability of pluripotent stem cells as a source for cell replacement therapies is currently being investigated, using farm animals as informative preclinical models. Disease modelling in farm animals allows systematic testing of effective treatments. Within the context of these developments, this concise review will focus on the contribution of farm animals to human health and welfare.

On ne peut traiter de la contribution des animaux d'élevage à la santé et au bienêtre de l'homme sans prendre en compte l'impact de la domestication des animaux sur la civilisation humaine. La révolution néolithique a commencé il y a environ 14 000 ans avec la domestication des animaux et des plantes, ce qui a donné naissance aux principales variétés cultivées et races d'élevage. En revanche, la sélection d'espèces animales nouvelles pour la recherche biomédicale, par exemple certaines espèces de petits rongeurs et d'autres modèles animaux, constitue une activité relativement récente. Ce n'est que depuis quelques dizaines d'années que les mécanismes cellulaires et moléculaires de l'hérédité sont bien compris et appliqués dans des approches permettant d'améliorer le potentiel génétique des élevages. Depuis quelques années, des découvertes fondamentales dans les domaines de la reprogrammation cellulaire, du génie génétique et du séquençage du génome entier ont accéléré cette évolution. Les premières protéines thérapeutiques produites par l'industrie biopharmaceutique chez des animaux d'élevage ont été approuvées pour traiter des patients humains. La recherche examine actuellement les possibilités de recourir à des cellules souches pluripotentes pour mettre en place des thérapies de remplacement, en utilisant des animaux d'élevage comme modèles précliniques. La modélisation des maladies en utilisant des animaux d'élevage permet d'effectuer des essais systématiques de l'efficacité des traitements. Les auteurs consacrent l'essentiel de leur synthèse à la contribution des animaux d'élevage à la santé et au bienêtre de l'homme, dans le cadre de ces évolutions.

No cabe examinar debidamente la contribución de los animales de granja a la salud y el bienestar del ser humano sin detenerse a reflexionar sobre la influencia que ha tenido en la civilización humana la domesticación de los animales. Hace unos 14 000 años, con la domesticación de animales y plantas, dio comienzo la revolución neolítica, que iba a deparar la aparición de las principales razas agrícolas de ganado y cultivos. En marcado contraste, la cría selectiva de nuevas especies animales con fines de investigación biomédica, como pequeños roedores y otras especies utilizadas como modelo, es una actividad relativamente reciente. Solo en los últimos decenios se han desentrañado los mecanismos celulares y moleculares de la herencia y se ha podido traducir este conocimiento en métodos para mejorar los niveles de éxito de la cría selectiva. En los últimos años, esta evolución se ha acelerado gracias a trascendentales descubrimientos en los ámbitos de la reprogramación celular, la ingeniería genética y la secuenciación de genomas completos. Ya están aprobadas las primeras proteínas terapéuticas para tratar a pacientes humanos obtenidas a partir de ganado mediante procedimientos biofarmacéuticos. Actualmente se investiga la idoneidad de las células troncales pluripotentes como fuente de terapias de sustitución celular, utilizando a animales de granja como modelos preclínicos informativos. La modelización de enfermedades en animales de granja permite ensayar tratamientos eficaces de forma sistemática. En el contexto de todos estos adelantos, los autores se centran en repasar concisamente la contribución de los animales de granja a la salud y el bienestar humanos.

siRNA mediated knockdown of tissue factor expression in pigs for xenotransplantation.

Acute vascular rejection (AVR), in particular microvascular thrombosis, is an important barrier to successful pig-to-primate xenotransplantation. Here, we report the generation of pigs with decreased tissue factor (TF) levels induced by small interfering (si)RNA-mediated gene silencing. Porcine fibroblasts were transfected with TF-targeting small hairpin (sh)RNA and used for somatic cell nuclear transfer. Offspring were analyzed for siRNA, TF mRNA and TF protein level. Functionality of TF downregulation was investigated by a whole blood clotting test and a flow chamber assay. TF siRNA was expressed in all twelve liveborn piglets. TF mRNA expression was reduced by 94.1 ± 4.7% in TF knockdown (TFkd) fibroblasts compared to wild-type (WT). TF protein expression in PAEC stimulated with 50 ng/mL TNF-α was significantly lower in TFkd pigs (mean fluorescence intensity TFkd: 7136 ± 136 vs. WT: 13 038 ± 1672). TF downregulation significantly increased clotting time (TFkd: 73.3 ± 8.8 min, WT: 45.8 ± 7.7 min, p < 0.0001) and significantly decreased thrombus formation compared to WT (mean thrombus coverage per viewing field in %; WT: 23.5 ± 13.0, TFkd: 2.6 ± 3.7, p < 0.0001). Our data show that a functional knockdown of TF is compatible with normal development and survival of pigs. TF knockdown could be a valuable component in the generation of multi-transgenic pigs for xenotransplantation.

Increased apoptosis in bovine blastocysts exposed to high levels of IGF1 is not associated with downregulation of the IGF1 receptor.

The hypothesis that high concentrations of IGF1 can impair embryo development was investigated in a bovine in vitro model to reflect conditions in polycystic ovary syndrome (PCOS) patients. Embryos were either cultured in the absence or presence of a physiological (100  ng/ml) or supraphysiological (1000  ng/ml) IGF1 concentration. Cell allocation, apoptosis, transcript and protein expression of selected genes involved in apoptosis, glucose metabolism and the IGF system were analysed. Supraphysiological IGF1 concentration did not improve blastocyst formation over controls, but induced higher levels of apoptosis, decreased TP53 protein expression in the trophectoderm and increased the number of cells in the inner cell mass (ICM). The increase in ICM cells corresponded with an increase in IGF1 receptor (IGF1R) protein in the ICM. A small, but significant, percentage of blastocysts displayed a hypertrophic ICM, not observed in controls and virtually absent in embryos treated with physiological concentrations of IGF1. Physiological IGF1 concentrations increased total IGF1R protein expression and upregulated IGFBP3 transcripts leading to an increase in blastocyst formation with no effects on cell number or apoptosis. In conclusion, the results support the hypothesis of detrimental effects of supraphysiological IGF1 concentrations on early pregnancy. However, our results do not support the premise that increased apoptosis associated with high levels of IGF1 is mediated via downregulation of the IGF1R as previously found in preimplantation mouse embryos. This in vitro system with the bovine preimplantation embryo reflects critical features of fertility in PCOS patients and could thus serve as a useful model for in-depth mechanistic studies.

Genome-wide expression profiling reveals distinct clusters of transcriptional regulation during bovine preimplantation development in vivo.

Bovine embryos can be generated by in vitro fertilization or somatic nuclear transfer; however, these differ from their in vivo counterparts in many aspects and exhibit a higher proportion of developmental abnormalities. Here, we determined for the first time the transcriptomes of bovine metaphase II oocytes and all stages of preimplantation embryos developing in vivo up to the blastocyst using the Affymetrix GeneChip Bovine Genome Array which examines approximately 23,000 transcripts. The data show that bovine oocytes and embryos transcribed a significantly higher number of genes than somatic cells. Several hundred genes were transcribed well before the 8-cell stage, at which the major activation of the bovine genome expression occurs. Importantly, stage-specific expression patterns in 2-cell, 4-cell, and 8-cell stages, and in morulae and blastocysts, were detected, indicating dynamic changes in the embryonic transcriptome and in groups of transiently active genes. Pathway analysis revealed >120 biochemical pathways that are operative in early preimplantation bovine development. Significant differences were observed between the mRNA expression profiles of in vivo and in vitro matured oocytes, highlighting the need to include in vivo derived oocytes/embryos in studies evaluating assisted reproductive techniques. This study provides the first comprehensive analysis of gene expression and transcriptome dynamics of in vivo developing bovine embryos and will serve as a basis for improving assisted reproductive technology.

Strategies for the derivation of pluripotent cells from farm animals.

Recent experiments demonstrated that forced expression of few critical genes drives conversion of a somatic into a pluripotent cell state. These induced pluripotent cells (iPS) were first generated from murine fibroblasts by Shinya Yamanaka's laboratory in 2006. By using retroviral vectors to express combinations of stemness genes, they identified Oct4, Sox2, Krueppel-like factor 4 and c-Myc as essential factors for reprogramming of somatic cells. Subsequent experiments applied this technology to human and rat fibroblasts, as well as other cell types and several groups showed that iPS can be generated by an even smaller number of transcription factors. The efficiency of conversion and maintenance of a pluripotent state can be supported by small molecules, such as valproic acid and specific pharmacological inhibitors. This technology is a milestone for a basic understanding of cell potency, cell fate and pathogenesis, as well as for development of cell therapies and potential applications in animal breeding.

Local neurotrophic repression of gene transcripts encoding fetal AChRs at rat neuromuscular synapses.

The spatio-temporal expression patterns of mRNA transcripts coding for acetylcholine receptor (AChR) subunits and myogenic factors were measured in denervated rat soleus muscle and in soleus muscle chronically paralyzed for up to 12 d by conduction block of the sciatic nerve by tetrodotoxin (TTX). In denervated muscle the AChR alpha-, beta-, gamma-, and delta-subunit mRNAs were elevated with highest expression levels in the former synaptic and the perisynaptic region and with lower levels in the extrasynaptic fiber segments. In muscle paralyzed by nerve conduction block the alpha-, beta-, gamma-, and delta-subunit mRNA levels increased only in extrasynaptic fiber segments. Surprisingly, in the synaptic region the gamma-subunit mRNA that specifies the fetal-type AChR, and alpha-, beta-, delta-subunit mRNAs were not elevated. The expression of the gene encoding the epsilon-subunit, which specifies the adult-type AChR, was always restricted to synaptic nuclei. The mRNA for the regulatory factor myogenin showed after denervation similar changes as the subunit transcripts of the fetal AChR. When the muscle was paralyzed by nerve conduction block the increase of myogenin transcripts was also less pronounced in synaptic regions compared to extrasynaptic fiber segments. The results suggest that in normal soleus muscle a neurotrophic signal from the nerve locally down-regulates the expression of fetal-type AChR channel in the synaptic and perisynaptic muscle membrane by inhibiting the expression of the gamma-subunit gene and that inhibition of the myogenin gene expression may contribute to this down-regulation.

Assessing Tn5 and Sleeping Beauty for transpositional transgenesis by cytoplasmic injection into bovine and ovine zygotes.

Transgenic domestic animals represent an alternative to bioreactors for large-scale production of biopharmaceuticals and could also provide more accurate biomedical models than rodents. However, their generation remains inefficient. Recently, DNA transposons allowed improved transgenesis efficiencies in mice and pigs. In this work, Tn5 and Sleeping Beauty (SB) transposon systems were evaluated for transgenesis by simple cytoplasmic injection in livestock zygotes. In the case of Tn5, the transposome complex of transposon nucleic acid and Tn5 protein was injected. In the case of SB, the supercoiled plasmids encoding a transposon and the SB transposase were co-injected. In vitro produced bovine zygotes were used to establish the cytoplasmic injection conditions. The in vitro cultured blastocysts were evaluated for reporter gene expression and genotyped. Subsequently, both transposon systems were injected in seasonally available ovine zygotes, employing transposons carrying the recombinant human factor IX driven by the beta-lactoglobulin promoter. The Tn5 approach did not result in transgenic lambs. In contrast, the Sleeping Beauty injection resulted in 2 lambs (29%) carrying the transgene. Both animals exhibited cellular mosaicism of the transgene. The extraembryonic tissues (placenta or umbilical cord) of three additional animals were also transgenic. These results show that transpositional transgenesis by cytoplasmic injection of SB transposon components can be applied for the production of transgenic lambs of pharmaceutical interest.

Isolation of bovine cardiomyocytes for reprogramming studies based on nuclear transfer.

The goal of this study was to establish and validate a protocol for preparing bovine cardiomyocytes from slaughterhouse material for nuclear transfer experiments. The cardiomyocyte was selected because it is a terminally differentiated cell and strongly expresses a unique subset of genes which can be monitored during the reprogramming period. A total of 39 trials were conducted, and an optimized protocol was developed yielding individual contractile cardiomyocytes from 3-5-month-old bovine fetuses The basic protocol involves stabilization of bovine heart tissue for transportation from the slaughterhouse to the laboratory by perfusion with Custodiol. This was followed by an enzymatic dissociation with collagenase in calcium-free medium and yielded individual contractile rod-shaped cardiomyocytes. Subsequent addition of Ca2+ caused the cardiomyocytes to round up which was an essential pre-condition for drawing them into glass transfer pipettes for delivery into the perivitelline space and for efficient electrofusion with cytoplasts derived from in vitro matured bovine oocytes. The use of cardiomyocytes maintained at 37 degrees C in nuclear transfer, resulted in a significantly reduced proportion of blastocysts compared to adult fibroblasts (14.0% versus 32.7%). Storage of cardiomyocytes at 4 degrees C prior to nuclear transfer was not compatible with blastocyst development. It is expected that this system will be valuable for investigating the reprogramming of gene expression which occurs after somatic cell nuclear transfer.

Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system.

The recently developed engineered nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) 9, provide new opportunities for gene editing in a straightforward manner. However, few reports are available regarding CRISPR application and efficiency in cattle. Here, the CRISPR/Cas9 system was used with the aim of inducing knockout and knock-in alleles of the bovine PRNP gene, responsible for mad cow disease, both in bovine fetal fibroblasts and in IVF embryos. Five single-guide RNAs were designed to target 875 bp of PRNP exon 3, and all five were codelivered with Cas9. The feasibility of inducing homologous recombination (HR) was evaluated with a reporter vector carrying EGFP flanked by 1 kbp PRNP regions (pHRegfp). For somatic cells, plasmids coding for Cas9 and for each of the five single-guide RNAs (pCMVCas9 and pSPgRNAs) were transfected under two different conditions (1X and 2X). For IVF zygotes, cytoplasmic injection was conducted with either plasmids or mRNA. For plasmid injection groups, 1 pg pCMVCas9 + 0.1 pg of each pSPgRNA (DNA2X) was used per zygote. In the case of RNA, two amounts (RNA1X and RNA2X) were compared. To assess the occurrence of HR, a group additionally cotransfected or coinjected with pHRegfp plasmid was included. Somatic cell lysates were analyzed by polymerase chain reaction and surveyor assay. In the case of embryos, the in vitro development and the genotype of blastocysts were evaluated by polymerase chain reaction and sequencing. In somatic cells, 2X transfection resulted in indels and large deletions of the targeted PRNP region. Regarding embryo injection, higher blastocyst rates were obtained for RNA injected groups (46/103 [44.6%] and 55/116 [47.4%] for RNA1X and RNA2X) than for the DNA2X group (26/140 [18.6%], P < 0.05). In 46% (26/56) of the total sequenced blastocysts, specific gene editing was detected. The total number of genetic modifications (29) was higher than the total number of gene-edited embryos, as three blastocysts from the group RNA2X reported more than one type of modification. The modifications included indels (10/56; 17.9%) and large deletions (19/56; 33.9%). Moreover, it was possible to detect HR in 1/8 (12.5%) embryos treated with RNA2X. These results report that the CRISPR/Cas9 system can be applied for site-specific edition of the bovine genome, which could have a great impact on the development of large animals resistant to important zoonotic diseases.

Cytomegalovirus early promoter induced expression of hCD59 in porcine organs provides protection against hyperacute rejection.

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.

Gene expression patterns in bovine in vitro-produced and nuclear transfer-derived embryos and their implications for early development.

Bovine in vitro-produced (IVP) and nuclear transfer (NT)-derived embryos differ from their in vivo-developed counterparts in a number of characteristics. A preeminent observation is the occurrence of the large offspring syndrome, which is correlated with considerable embryonic fetal and postnatal losses. We summarize here results from our studies in which we compared gene expression patterns from IVP and NT-derived embryos with those from their IVP counterparts. Numerous aberrations were found in IVP and NT-derived embryos, including a complete lack of expression, an induced expression, or a significant up- or downregulation of a specific gene. These alterations may affect a number of physiological functions and are considered as a kind of stress response of the embryos to deficient environmental conditions. We hypothesize that the alterations are caused by epigenetic modifications, primarily by changes in the methylation patterns. Unravelling these epigenetic modifications is promising to reveal the underlying mechanisms of the large offspring syndrome.

Transgenic livestock: premises and promises.

Microinjection of DNA constructs into pronuclei of zygotes has been the method of choice for the generation of transgenic livestock. However, this procedure is characterized by low efficiency (1-4% transgenic offspring), random integration and variable expression of the transgene as well as a considerable proportion of mosaicism. Furthermore, it is extremely time consuming and costly. As a consequence, commercial application has focused on the production of recombinant proteins in the mammary gland of transgenic animals and xenotransplantation, e.g. the use of porcine organs in human organ transplantation. In addition, transgenic pigs carrying a modified porcine growth hormone (hMt-pGH) construct show significant improvements in economically important traits without adverse side effects of a GH overproduction. Improvements of transgenic technology will likely come from the generation of appropriate cell lines suitable for transfection or even homologous recombination and their subsequent use in nuclear transfer. Additionally, in the mouse a number of sophisticated molecular tools have been developed that allow precise modifications of the genome. These include the application of artificial chromosomes from yeast (YAC) or bacteria (BAC) for position-independent and copy-number-dependent expression of a transgene, the Tet-system (tetracycline inducible) for a tight temporal control of transgene expression, as well as conditional mutagenesis by applying site-specific DNA recombinases (e.g. Cre, FLP). The successful adaptation of these molecular tools to livestock will enable the fulfillment of many of the promises originally thought to be achievable when transgenic livestock were first reported.

In vivo oocyte IGF-1 priming increases inner cell mass proliferation of in vitro-formed bovine blastocysts.

Studies addressing the effects of supraphysiological levels of IGF-1 on oocyte developmental competence are relevant for unravelling conditions resulting in high bioavailability of IGF-1, such as the polycystic ovary syndrome (PCOS). This study investigated the effects of supraphysiological levels of IGF-1 during in vivo folliculogenesis on the morula-blastocyst transition in bovine embryos. Compacted morulae were non-surgically collected and frozen for subsequent mRNA expression analysis (IGF1R, IGBP3, TP53, AKT1, SLC2A1, SLC2A3, and SLC2A8), or underwent confocal microscopy analysis for protein localization (IGF1R and TP53), or were cultured in vitro for 24 h. In vitro-formed blastocysts were subjected to differential cell staining. The mRNA expression of SLC2A8 was higher in morulae collected from cows treated with IGF-1. Both IGF1R and TP53 protein were present in the plasma membrane and cytoplasm. IGF-1 treatment did not affect protein localization of both IGF1R and TP53. In vitro-formed blastocysts derived from morulae recovered from IGF-1-treated cows displayed a higher number of cells in the inner cell mass (ICM). Total cell number (TCN) of in vitro-formed blastocysts was not affected. A higher mean ICM/TCN proportion was observed in in vitro-formed blastocysts derived from morulae collected from cows treated with IGF-1. The percentage of in vitro-formed blastocysts displaying a low ICM/TCN proportion was decreased by IGF-1 treatment. In vitro-formed blastocysts with a high ICM/TCN proportion were only detected in IGF-1 treated cows. Results show that even a short in vivo exposure of oocytes to a supraphysiological IGF-1 microenvironment can increase ICM cell proliferation in vitro during the morula to blastocyst transition.

Inhibition of porcine endogenous retroviruses (PERVs) in primary porcine cells by RNA interference using lentiviral vectors.

A potential risk in pig-to-human xenotransplantation is the transmission of PERVs to human recipients. Here we show for the first time the inhibition of PERV expression in primary porcine cells by RNA interference using lentiviral vectors. Cells were transduced with lentiviral vectors coding for short hairpin (sh) RNAs directed against PERV. In all primary porcine cells studied and in the porcine kidney cell line PK-15, expression of PERV-mRNA was significantly reduced as measured by real-time PCR. Most importantly, expression of PERV proteins was almost completely suppressed, as shown by Western blot analysis. Thus, lentiviral shRNA vectors could be used to knockdown PERV expression and create transgenic pigs with a reduced risk of PERV transmission during xenotransplantation.

High incidence of single nucleotide polymorphisms in the prion protein gene of native Brazilian Caracu cattle.

Different alleles of the human and ovine prion protein gene correlate with a varying susceptibility to transmissible spongiform encephalopathies. However, the pathogenic implications of specific polymorphisms in the bovine prion protein gene (PRNP) are only poorly understood. Previous studies on the bovine PRNP gene investigated common European and North American cattle breeds. As a consequence of decades of intensive breeding for specific traits, these modern breeds represent only a small fraction of the bovine gene pool. In this study, we analysed PRNP polymorphisms in the native Brazilian Caracu breed, which developed in geographical isolation since the 16th century. A total of 10 single nucleotide polymorphisms (SNPs) were discovered in the coding region of the Caracu PRNP gene. Eight of the SNPs occurred at high frequencies in Caracu cattle (variant allele frequencies = 0.10-0.76), but were absent or only rarely observed in European and North American breeds. One of the Caracu SNPs was associated with an amino acid exchange from serine to asparagine (f = 0.17). This SNP was not detected in Holstein-Friesian, Simmental and German Gelbvieh and was only rarely detected in beef cattle (f = 0.01). We found 17 haplotypes for PRNP in the Caracu breed.

Differential expression patterns of five acetylcholine receptor subunit genes in rat muscle during development.

The spatial and temporal expression patterns of five genes which encode the alpha-, beta-, gamma-, delta- and epsilon-subunits of the nicotinic acetylcholine receptor in skeletal muscle were followed during development in the rat by in situ hybridization analysis. Three major developmental phases, characterized by specific expression patterns, could be distinguished. (i) During myogenic differentiation alpha-, beta-, gamma- and delta-subunit genes are activated and transcripts are expressed in muscle precursor cells at embryonic day 12 (E12) and during subsequent cell fusion. (ii) Following innervation of myotubes at approximately E15-E17 the mRNA of the alpha-, beta-, gamma- and delta-subunit genes accumulate in synaptic and decrease in extrasynaptic fibre regions during early synaptogenesis. The mRNA of the epsilon-subunit gene becomes detectable first in subsynaptic nuclei 2-3 days after innervation has occurred. (iii) During postnatal development alpha-, beta- and delta- subunit transcript levels are reduced predominantly in extrasynaptic fibre segments and show significant differences in distribution depending on the muscle subtype whereas the gamma-subunit mRNA disappears completely within the first postnatal week in all muscles. In contrast, the epsilon-subunit gene is transcribed only in subsynaptic myonuclei throughout development and in the adult muscle.

Cell cycle synchronization of porcine fetal fibroblasts: effects of serum deprivation and reversible cell cycle inhibitors.

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.

Nerve-dependent induction of AChR epsilon-subunit gene expression in muscle is independent of state of differentiation.

To determine if the expression of the epsilon-subunit of the acetylcholine receptor by the subsynaptic nuclei in skeletal muscle is dependent on the state of differentiation of the muscle, we have compared the spatiotemporal distribution of epsilon-subunit transcripts during synapse formation in fetal and adult muscle. Both during "ontogenic" synaptogenesis in the fetus and during "ectopic" synaptogenesis in the adult animal the motor nerve induced focally the expression of the epsilon-subunit mRNA in subsynaptic nuclei. The temporal expression patterns at both types of developing synapses were similar. The results support the view that in muscle developing in vivo epsilon-subunit gene transcription and its stabilization in subsynaptic nuclei is exclusively controlled by the motor neuron, independently of the developmental state of the muscle nuclei. Thus, both nerve and muscle remain plastic in their respective abilities to induce and express the synapse-specific combination of AChR subunit genes.

Increased gene expression of acetylcholine receptor and myogenic factors in passively transferred experimental autoimmune myasthenia gravis.

The passive transfer of myasthenia gravis by injection of mAb against muscle acetylcholine receptor (AChR) alpha-subunit, results in increased expression of AChR subunit genes, mainly at synaptic regions. The gene expression of AChR and of other muscle-specific proteins is regulated in a similar manner in passively transferred experimental autoimmune myasthenia gravis (EAMG) and in AChR-induced EAMG. Administration of AChR-specific mAb leads to a significant reduction in muscle AChR content and to an elevation in the mRNA levels corresponding to the adult, synaptic type of the receptor, as shown by Northern blot and in situ hybridization analyses. The mRNA levels of the myogenic factors myogenin and MRF4 are also increased moderately, whereas MyoD transcript levels remain unchanged. Thus, passive transfer of EAMG by mAb directed to defined epitopes of AChR alpha-subunit provides a suitable model for analyzing and following the cascade of molecular events triggered by anti-AChR immunopathologic antibodies and may shed light on the regulatory mechanisms underlying the human disease as well.