Transgenic Fetuses Research Articles

Cyclophilin A as a Pro-Inflammatory Factor Exhibits Embryotoxic and Teratogenic Effects during Fetal Organogenesis

The precise balance of Th1, Th2, and Th17 cytokines is a key factor in successful pregnancy and normal embryonic development. However, to date, not all humoral factors that regulate and influence physiological pregnancy have been completely studied. Our data here pointed out cyclophilin A (CypA) as the adverse pro-inflammatory factor negatively affecting fetal development and associated with pregnancy complications. In different mouse models in vivo, we demonstrated dramatic embryotoxicity and teratogenicity of increased CypA levels during pregnancy. Using generated transgenic models, we showed that CypA overexpression in fetal tissues induced the death of all transgenic fetuses and complete miscarriage. Administration of recombinant human CypA in a high dose to pregnant females during fetal organogenesis (6.5–11.5 dpc) exhibited teratogenic effects, causing severe defects in the brain and bone development that could lead to malformations and postnatal behavioral and cognitive disorders in the offspring. Embryotoxic and teratogenic effects could be mediated by CypA-induced up-regulation of M1 macrophage polarization via activation of the STAT1/3 signaling pathways. Here, we propose secreted CypA as a novel marker of complicated pregnancy and a therapeutic target for the correction of pregnancy complications.

Pregnancy-Induced High Plasma Levels of Soluble Endoglin in Mice Lead to Preeclampsia Symptoms and Placental Abnormalities

Preeclampsia is a pregnancy-specific disease of high prevalence characterized by the onset of hypertension, among other maternal or fetal signs. Its etiopathogenesis remains elusive, but it is widely accepted that abnormal placentation results in the release of soluble factors that cause the clinical manifestations of the disease. An increased level of soluble endoglin (sEng) in plasma has been proposed to be an early diagnostic and prognostic biomarker of this disease. A pathogenic function of sEng involving hypertension has also been reported in several animal models with high levels of plasma sEng not directly dependent on pregnancy. The aim of this work was to study the functional effect of high plasma levels of sEng in the pathophysiology of preeclampsia in a model of pregnant mice, in which the levels of sEng in the maternal blood during pregnancy replicate the conditions of human preeclampsia. Our results show that wild type pregnant mice carrying human sEng-expressing transgenic fetuses (fWT(hsEng+)) present high plasma levels of sEng with a timing profile similar to that of human preeclampsia. High plasma levels of human sEng (hsEng) are associated with hypertension, proteinuria, fetal growth restriction, and the release of soluble factors to maternal plasma. In addition, fWT(hsEng+) mice also present placental alterations comparable to those caused by the poor remodeling of the spiral arteries characteristic of preeclampsia. In vitro and ex vivo experiments, performed in a human trophoblast cell line and human placental explants, show that sEng interferes with trophoblast invasion and the associated pseudovasculogenesis, a process by which cytotrophoblasts switch from an epithelial to an endothelial phenotype, both events being related to remodeling of the spiral arteries. Our findings provide a novel and useful animal model for future research in preeclampsia and reveal a much more relevant role of sEng in preeclampsia than initially proposed.

Hydrodynamics-Based Transplacental Delivery as a Useful Noninvasive Tool for Manipulating Fetal Genome.

We previously demonstrated that the injection of pregnant wild-type female mice (carrying enhanced green fluorescent protein (EGFP)-expressing transgenic fetuses) at embryonic day (E) 12.5 with an all-in-one plasmid conferring the expression of both Cas9 and guide RNA (targeted to the EGFP cDNA) complexed with the gene delivery reagent, resulted in some fetuses exhibiting reduced fluorescence in their hearts and gene insertion/deletion (indel) mutations. In this study, we examined whether the endogenous myosin heavy-chain α (MHCα) gene can be successfully genome-edited by this method in the absence of a gene delivery reagent with potential fetal toxicity. For this, we employed a hydrodynamics-based gene delivery (HGD) system with the aim of ensuring fetal gene delivery rates and biosafety. We also investigated which embryonic stages are suitable for the induction of genome editing in fetuses. Of the three pregnant females injected at E9.5, one had mutated fetuses: all examined fetuses carried exogenous plasmid DNA, and four of 10 (40%) exhibited mosaic indel mutations in MHCα. Gene delivery to fetuses at E12.5 and E15.5 did not cause mutations. Thus, the HGD-based transplacental delivery of a genome editing vector may be able to manipulate the fetal genomes of E9.5 fetuses.

Comparative Transcriptome Analysis Provides Insights into the Polyunsaturated Fatty Acid Synthesis Regulation of Fat-1 Transgenic Sheep.

Transgenic technology has huge application potential in agriculture and medical fields, such as producing new livestock varieties with new valuable features and xenotransplantation. However, how an exogenous gene affects the host animal’s gene regulation networks and their health status is still poorly understood. In the current study, Fat-1 transgenic sheep were generated, and the tissues from 100-day abnormal (DAF_1) and normal (DAF_2) fetuses, postnatal lambs (DAF_4), transgenic-silencing (DAFG5), and -expressing (DAFG6) skin cells were collected and subjected to transcriptome sequencing, and their gene expression profiles were compared in multiple dimensions. The results were as follows. For DAF_1, its abnormal development was caused by pathogen invasion but not the introduction of the Fat-1 gene. Fat-1 expression down-regulated the genes related to the cell cycle; the NF-κB signaling pathway and the PI3K/Akt signaling pathway were down-regulated, and the PUFAs (polyunsaturated fatty acids) biosynthesis pathway was shifted toward the biosynthesis of high-level n-3 LC-PUFAs (long-chain PUFAs). Four key node genes, FADS2, PPARA, PRKACA, and ACACA, were found to be responsible for the gene expression profile shift from the Fat-1 transgenic 100-day fetus to postnatal lamb, and FADS2 may play a key role in the accumulation of n-3 LC-PUFAs in Fat-1 transgenic sheep muscle. Our study provides new insights into the FUFAs synthesis regulation in Fat-1 transgenic animals.

Gradual differentiation and confinement of the cardiac conduction system as indicated by marker gene expression

The components of the cardiac conduction system, responsible for coordinated activation of the heart chambers, are well defined and their cells differ in gene expression profile and phenotype from those of the surrounding working myocardium. Yet, when and on what basis the myocardium of each of the conduction system components become distinguishable from other myocardium during heart development has not been well established. To identify and assess cell type-specific expression profiles and differentiation markers, we performed transcriptome analysis on fluorescence activated cell sorted purified conduction system (Venus+) and chamber myocardial cells (Katushka+) of Tbx3+/Venus;TgNppb(Katushka) double transgenic mouse fetuses. We found that transcripts associated with nervous system development and ion channel activity were enriched in Tbx3+ conduction system cells, whereas transcripts associated with mitochondrial function, muscle contraction and fatty acid metabolism were enriched in the Nppb+ working myocardium. We analyzed spatio-temporal expression patterns of several candidate markers (Cacna2d2, Cacna1g, Ephb3, Tnni1), reviewed those of established conduction system markers (Tbx3, Hcn4, Gja5, Cntn2), and placed the patterns in the context of conduction system development. The overview indicates that different properties of conduction system components develop gradually and at different developmental stages, and that chamber myocardium gradually differentiates and diverges from conduction system myocardium until after birth.

Quantitative evaluation on 3D fetus morphology via X‐ray grating based imaging technique

AbstractSynchrotron‐based X‐ray phase sensitive micro‐tomography techniques enable to visualize detailed three‐dimensional (3D) insight into nondestructive inner‐structure of biomedical samples. Different phase sensitive mechanisms have been employed for discrimination of tissue's tiny density variations in biomedical research. We effectively visualized and analyzed the phase‐contrast experimental results of X‐ray grating‐based imaging, based on grating interferometry with phase stepping, by using transgenic mouse fetus. We quantitatively measured and evaluated the contrast‐to‐noise ratio or the mass density resolution, spatial resolution, radiation dose, and figure of merit of X‐ray grating‐based imaging technique in biomedical research respectively. Moreover, the complex coherent degrees of light source were duly taken into account in the analysis of spatial resolution. In addition, the mass density distribution of soft biomedical specimens can be estimated using our presented method preliminarily. For most soft tissue and organ observation, this work provides explicit guidelines to help future synchrotron users obtain the quantitative image information, suitable for their specific biomedical research.

63. Molecular and functional long-term effects of preeclampsia on the cardiovascular system assessed on a mouse model of severe preeclampsia

Preeclampsia (PE) is a characterized by de novo hypertension and proteinuria. Women affected with PE have an increased risk of chronic hypertension and premature cardiovascular disease years later. Mice bearing transgenic fetuses overexpressing the human transcription factor STOX1 (storkhead box1) develop a severe preeclamptic phenotype. The molecular analysis of long term cardiovascular risks induced by preeclampsia will be studied using our mouse model. We started with 10 eight to ten-months-old mice, 5 had control gestations and 5 preeclamptic gestations 6 to 8 months earlier. We analyzed heart functional parameters by ultrasonography, in normal and stressed conditions. Then we carried out a transcriptional profile of endothelial cells and hearts of the mice. Fibrosis was evaluated by a histology analysis of the hearts. Finally the cytokine levels in mice plasma will be analyzed on 33 cytokines. The relative mass of the heart of the preeclamptic mice was increased by 11% (p = 0.017), with an important fibrosis. Ultrasonography revealed a lesser morphological adaptation to stress. Under dobutamin aortic peak velocity, blood pressure, and right ventricular outflow are significantly increased. The endothelial cells microarray analysis demonstrate that ∼3073 transcripts (p Our results demonstrate massive quasi-invisible long term effects of preeclampsia, affecting strongly the endothelial cells. Dilatation of hearts and fibrosis indicate a noxious cardiac tissue remodeling in preeclamptic mice. To the best of our knowledge, our study is one of the first to address the molecular effects of preeclampsia in the long term.

Overexpressing dominant-negative FGFR2-IIIb impedes lung branching morphogenesis in pigs

Genetic studies with mouse models have shown that fibroblast growth factor receptor 2-IIIb (FGFR2-IIIb) plays crucial roles in lung development and differentiation. To evaluate the effect of FGFR2-IIIb in pig lung development, we employed somatic cell nuclear transfer (SCNT) technology to generate transgenic pig fetuses overexpressing the transmembrane (dnFGFR2-IIIb-Tm) and soluble (dnFGFR2-IIIb-HFc) forms of the dominant-negative human FGFR2-IIIb driven by the human surfactant protein C (SP-C) promoter, which was specifically expressed in lung epithelia. Eight dnFGFR2-IIIb-Tm transgenic and twelve dnFGFR2-IIIb-HFc transgenic pig fetuses were collected from three and two recipient sows, respectively. Repression of FGFR2-IIIb in lung epithelia resulted in smaller lobes and retardation of alveolarization in both forms of dnFGFR2-IIIb transgenic fetuses. Moreover, the dnFGFR2-IIIb-HFc transgenic ones showed more deterioration in lung development. Our results demonstrate that disruption of FGFR2-IIIb signaling in the epithelium impedes normal branching and alveolarization in pig lungs, which is less severe than the results observed in transgenic mice. The dnFGFR2-IIIb transgenic pig is a good model for the studies of blastocyst complementation as well as the mechanisms of lung development and organogenesis.

Differentiation of Induced Pluripotent Stem Cells to Lentoid Bodies Expressing a Lens Cell-Specific Fluorescent Reporter.

Curative approaches for eye cataracts and other eye abnormalities, such as myopia and hyperopia currently suffer from a lack of appropriate models. Here, we present a new approach for in vitro growth of lentoid bodies from induced pluripotent stem (iPS) cells as a tool for ophthalmological research. We generated a transgenic mouse line with lens-specific expression of a fluorescent reporter driven by the alphaA crystallin promoter. Fetal fibroblasts were isolated from transgenic fetuses, reprogrammed to iPS cells, and differentiated to lentoid bodies exploiting the specific fluorescence of the lens cell-specific reporter. The employment of cell type-specific reporters for establishing and optimizing differentiation in vitro seems to be an efficient and generally applicable approach for developing differentiation protocols for desired cell populations.

357 EARLY FETAL DEVELOPMENT OF NUCLEAR TRANSFER BOVINE EMBRYOS GENERATED FROM FIBROBLASTS GENETICALLY MODIFIED BY piggyBac TRANSPOSITION

Transposon-mediated transgenesis is a well-established tool for genome manipulation in small animal models. However, translation of this active transgenesis method to the large animal setting requires further investigation. We have previously demonstrated that a helper-independent piggyBac (PB) transposon system can efficiently transpose transgenes into the bovine genome [Alessio et al. 2014 Reprod. Domest. Anim. 49 (Suppl. 1), 8]. The aims of the current study were a) to investigate the effectiveness of a hyperactive version of the PB transposase, and b) to determine the ability of the genetically modified cells to support early embryo and fetal development upon somatic cell nuclear transfer (SCNT). Bovine fetal fibroblasts (BFF) were chemically transfected with either pmGENIE-3 (a helper-independent PB transposon conferring genes for hygromycin resistance and enhanced green fluorescent protein (EGFP); Urschitz et al. 2010 PNAS USA 107, 8117–8122), pmhyGENIE-3 (carrying an hyperactive version of the PB transposase; Marh et al. 2012 PNAS USA 109, 19 184–19 189), or pmGENIE-3/Δ PB (a control plasmid lacking a functional PB transposase). Upon transfection, cell cultures were subjected to 14 days of hygromycin selection. Antibiotic-resistant and EGFP+ colonies were counted and data analysed by ANOVA and Tukey's test. For SCNT, pmhyGENIE-3 and pmGENIE-3 polyclonal cell lines were selected by FACS and individual cells used as nuclear donors. Day 7 blastocysts were nonsurgically transferred to synchronized recipients. Conceptuses were recovered by Day 35 of gestation, observed under fluorescence excitation, and genotyped. The mean number of colonies in pmhyGENIE-3 group was significantly higher than those in pmGENIE-3 and the control group (324.0 ± 17.8 v. 100.0 ± 16.1 and 2.8 ± 0.8 respectively, n = 4–7; P < 0.05). The hyperactive transposase increased transgene integration efficiency 3.24 times compared with the conventional PB transposase. The SCNT and early fetal development data are summarised in Table 1. Phenotypic analysis revealed that both transgenic fetuses and the extraembryonic membranes expressed EGFP with no macroscopic evidence of variegated transgene expression. Molecular analysis by PCR confirmed that both fetuses carried the transposon DNA. Here, we demonstrate that a hyperactive version of the PB transposase is more active in bovine cells than the conventional PB transposase. In addition, SCNT embryos generated from genetically modified cells by the pGENIE transposon system can progress to early stages of fetal development. Table 1.SCNT and early fetal development of bovine fibroblasts transposed with piggyBac1 The financial support of UNRC, CONICET and ANPCyT from Argentina is gratefully acknowledged.

328 NONVIRAL REPROGRAMMING OF mCHERRY-EXPRESSING PORCINE FIBROBLASTS INTO INDUCED PLURIPOTENT STEM CELLS BY piggyBac TRANSPOSONS

The generation of induced pluripotent stem (iPS) cells is a promising approach for innovative cell therapies, as well as for animal biotechnology. The original method requires viral transduction of several reprogramming factors, which may be associated with an increased risk of tumorigenicity due to the preferential integration into active genes. The domestic pig is an attractive large animal model for preclinical testing of safety and efficacy of cell-based therapies. Porcine organs are similar in size and physiology to their human counterparts, and a suitable model for cardiovascular disease, muscular dystrophies, atherosclerosis, wound repair, diabetes, and ophthalmological diseases. Therefore, the present study was carried out to derive porcine iPS cells from transgenic fetuses systemically expressing mCherry (Garrels et al. 2011 PLOS ONE 6) through a nonviral piggyBac transposon. The piggyBac transposon system has several advantages: (i) piggyBac has no bias to integrate in expressed gene-like lenti- or retroviral vectors, (ii) the cargo capacity is >100 kb, (iii) seamless removal is possible, and (iv) the production of transposon plasmid is cost-efficient and does not require S2 safety cabinets. Porcine fetal fibroblasts isolated from CAGGS-mCherry founder porcine line fetuses (passage 2), were co-electroporated with a PB transposon carrying a multigene cassette consisting of human cDNA for OCT4, SOX2, KLF4, c-MYc, NANOG, and LIN28 separated by self-cleaving 2A peptide sequences, driven by a CAGGS promoter and a helper plasmid expressing the pCMV-PB transposase. On Day 6 postelectroporation, morphology of fibroblasts started change to round structure, and on Day 9 loose aggregates of cells developed. Putative iPS cell colonies were cultured, propagated, and characterised through morphology and expression of pluripotency markers, such as AP, OCT4, SSEA-1, and SSEA-4, through immunostaining. Further, various stemness genes, including OCT4, SOX2, NANOG, and UTF, were detected by porcine-specific primers through endpoint RT-PCR. In vitro differentiation potential was assessed by embryoid body (EB) formation. The formed EB exhibited the expression of mCherry in their cells and expressed differentiation markers, such as NESTIN, TUJI, GATA4 and AFP. To test their tumorigenic potential, 1 × 106 iPS cells were injected under the skin of nude mice. An mCherry-positive tumour was recovered 6 weeks later. Presently the tumour is being prepared for histological analysis. This study indicates that piggyBac transposon containing 6 transcription factors is able to reprogram porcine fetal fibroblasts into iPS cells. These cells could be cultured and maintained in vitro for a prolonged period, exhibit characteristics of stem cells, and offer a potential source for future blastocyst complementation experiments.

Biological characteristics of cleft palate relevant gene thyroid transcription factor-2 transgenic mice

The aim of this study is to establish a transgenic mouse model for cleft palate relevant gene thyroid transcription factor-2 (TTF-2), which can be used to study palatal shelf development when the expression pattern and regular activation of TTF-2 is altered. The C57BL/6J mouse TTF-2 gene was cloned through polymerase chain reaction (PCR) from the mouse genomic DNA. The TTF-2 gene was inserted into the expression vector pBROAD3-mcs to construct the recombinant expression vector pBROAD3-TTF-2. This expression vector was then microinjected into the male pronuclei of the fertilized mouse ovum. Thus, the TTF-2 transgenic mice model was established. The genotype of the transgenic mice was identified by PCR and Southern blot analysis. Immunohistochemistry identified the consistent expression of TTF-2 gene during its palatal shelf development. TTF-2 genes were microinjected into 982 fertilized ova. A total of 580 two-cell-stage embryos cultured and transplanted into the oviducts of 48 pseudopregnant female mice. Overall, 68 embryos were obtained for analysis. The genotype of the mice was determined through PCR and Southern blot analysis using genomic DNA extracted from tail biopsies of the transgenic fetus. A total of 13 TTF-2 transgenic mice were detected. The expression of TTF-2 gene during the palatal shelf development of the transgenic mice was consistently detected by immunohistochemistry. The recombinant expression vector pBROAD3-TTF-2 was integrated into mouse genome through microinjection. The transgenic mouse in the palatal shelf that consistently expressed TTF-2 was successfully established and displayed a cleft palate phenotype.

Ultrasonographic evaluation of hG-CSF transgenic goat conceptus

The objective of this study was to evaluate the development of transgenic (T) goat embryos and fetuses for human Granulocyte Colony Stimulating Factor (hG-CSF) by ultrasonography. Four pregnancies in non-transgenic (NT) goats were obtained after fertilization (either fixed-time artificial insemination or natural mating) using the T male for hG-CSF. Ultrasound examinations were carried out at 30, 40 (transrectal via), 50, 60, 90 and 120 days of pregnancy (transabdominal via). Some parameters were observed such as morphology, organogenesis and formation of skeletal fetuses, viability with cardiac activity and fetuses’ movements. Measurements were taken of the crown-rump length, diameter of embryonic vesicle, thorax, abdomen, umbilical cord and placentomes. After parturition, DNA testing was conducted in all offspring and 4 T and 2 NT kids were identified. The conceptus started their differentiation at 40 days. The heart was detected in all examinations and the heart chambers were assessed at 50 days. Gastric compartments, liver and kidneys were observed at 60 days, the same period that all bony structures were visualized. Average values ​​of all evaluated parameters had a gradual increase with the progression of pregnancy. T and NT goat embryos and fetuses had a similar growth and all remained viable throughout the experimental period.

Bovine fetal epithelium cells expressing shRNA targeting viral VP1 gene resisted against foot-and-mouth disease virus

RNAi could protect experimental animals from Foot-and-mouth disease virus (FMDV), but pivotal issue is delivery of RNAi. In this study, shRNA recombinant lentiviral plasmid RNAi-LT6 targeting VP1 of FMDV, which strongly suppressed the transient expression of a FLAG-tagged VP1 protein in 293T cells and significantly inhibited viral replication in BHK-21 cells, was screened and transfected into bovine fetal fibroblast cells. With subsequent somatic cell cloning, three 4-month-old transgenic fetuses were obtained, and integration into chromosome and expression of shRNA in primary transgenic tongue epithelium cells were confirmed by Southern hybridization and Northern assay, respectively. shRNA significantly suppressed viral RNA synthesis and viral replication in primary transgenic tongue epithelium cells after inoculation of 200 TCID50 of FMDV. The results suggested that transgenic cloning may be a useful tool for RNAi delivery and RNAi anti-viral strategy, and RNAi-LT6 could be a candidate shRNA used for preparation of transgenic cattle against FMDV.

327 PRODUCTION OF TRIPLE TRANSGENIC hHO-1/GGTA-1–/–/hCD55 TRANSGENIC PIGS USING SLEEPING BEAUTY TRANSPOSITION AND ZINC-FINGER NUCLEASES

Advances in xenotransplantation (pig-to-baboon or human transplantation) require multi-transgenic pigs with a homozygous knockout (KO) of the α1,3-galactosyltransferase gene (GGTA-1, encoding for Gal-epitopes) to control the hyperacute rejection response. To achieve prolonged survival of the porcine xenograft, transgenic expression of additional immune modulatory genes on the GGTA-1–/– background is considered the solution of choice. The acute vascular rejection (AVR) is primarily caused by endothelial cell activation and leads to rejection of GGTA-1–/– pig organs. Here, we set out to produce triple transgenic pigs with high expression of the human complement regulatory gene hCD55 (human decay acceleration factor, hDAF) and the anti-apoptotic and anti-inflammatory gene human heme oxygenase-1 (hHO-1), respectively. Porcine ear fibroblasts carrying a hHO-1 transgene (Petersen et al. 2011 Xenotransplantation 18, 355–368) were transfected with zinc-finger nucleases (ZFN) targeting the GGTA-1 gene, leading to a biallelic KO of the GGTA-1 gene in ~1% of the transfected cells (Hauschild et al. 2011 Proc. Natl. Acad. Sci. USA 108, 15 010). Somatic cell nuclear transfer (SCNT) was accomplished with cells from double transgenic cell line (hHO-1/GGTA-1–/–), and one pregnancy was terminated to obtain fetal cell cultures. Fluorescence-activated cell sorting (FACS) analysis revealed that all 11 fetuses (C6F1-F11) were lacking Gal-epitopes. One pure fetal cell culture (C6F5) was used for co-transfection with the newly designed SB-CAGGS-hDAF-Puror transposon, based on the Sleeping Beauty transposon plasmid pT2/HB, together with the SB transposase 100X plasmid (both kindly provided by Dr. Zoltan Ivics). After 14 days of selection with puromycin, cell colonies were picked and expanded to obtain cells for analysis and as a backup for SCNT. The PCR amplification with hDAF-specific primers revealed that ~50% of picked colonies had integrated the transgene. Two hDAF-positive colonies were pooled and used for SCNT. One pregnancy was sacrificed and 4 hHO-1/GGTA-1–/–/hCD55 fetuses were obtained for real-time PCR analysis and subsequent use in SCNT. Real-time PCR showed elevated hCD55 expression representing a 0.9-fold (0.7- to 1.0-fold) expression of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH), while FACS analysis confirmed absence of Gal-epitopes in all 4 fetuses. Binding of human-specific anti-CD55 antibodies showed high expression of hCD55 by FACS measurement. Triple transgenic fetuses C8F2 and C8F4 showed higher expression of hCD55 by real-time PCR and by FACS analysis compared with fetuses C8F1 and C8F3. Fetuses C8F3 and C8F4 were used for re-cloning experiments to produce live offspring, which will further be characterised and used in transplantation experiments. This work underlines the importance of new genomic technologies to further improve the efficiency of the generation of transgenic pigs suitable for xenotransplantation.

Identification of Short Hairpin RNA Targeting Foot-And-Mouth Disease Virus with Transgenic Bovine Fetal Epithelium Cells

BackgroundAlthough it is known that RNA interference (RNAi) targeting viral genes protects experimental animals, such as mice, from the challenge of Foot-and-mouth disease virus (FMDV), it has not been previously investigated whether shRNAs targeting FMDV in transgenic dairy cattle or primary transgenic bovine epithelium cells will confer resistance against FMDV challenge.Principal FindingHere we constructed three recombinant lentiviral vectors containing shRNA against VP2 (RNAi-VP2), VP3 (RNAi-VP3), or VP4 (RNAi-VP4) of FMDV, and found that all of them strongly suppressed the transient expression of a FLAG-tagged viral gene fusion protein in 293T cells. In BHK-21 cells, RNAi-VP4 was found to be more potent in inhibition of viral replication than the others with over 98% inhibition of viral replication. Therefore, recombinant lentiviral vector RNAi-VP4 was transfected into bovine fetal fibroblast cells to generate transgenic nuclear donor cells. With subsequent somatic cell cloning, we generated forty transgenic blastocysts, and then transferred them to 20 synchronized recipient cows. Three transgenic bovine fetuses were obtained after pregnant period of 4 months, and integration into chromosome in cloned fetuses was confirmed by Southern hybridization. The primary tongue epithelium cells of transgenic fetuses were isolated and inoculated with 100 TCID50 of FMDV, and it was observed that shRNA significantly suppressed viral RNA synthesis and inhibited over 91% of viral replication after inoculation of FMDV for 48 h.ConclusionRNAi-VP4 targeting viral VP4 gene appears to prevent primary epithelium cells of transgenic bovine fetus from FMDV infection, and it could be a candidate shRNA used for cultivation of transgenic cattle against FMDV.

Somatic Cell Nuclear Transfer Cloning: Practical Applications and Current Legislation

Somatic cloning is emerging as a new biotechnology by which the opportunities arising from the advances in molecular genetics and genome analysis can be implemented in animal breeding. Significant improvements have been made in SCNT protocols in the past years which now allow to embarking on practical applications. The main areas of application of SCNT are: Reproductive cloning, therapeutic cloning and basic research. A great application potential of SCNT based cloning is the production of genetically modified (transgenic) animals. Somatic cell nuclear transfer based transgenic animal production has significant advances over the previously employed microinjection of foreign DNA into pronuclei of zygotes. This cell based transgenesis is compatible with gene targeting and allows both, the addition of a specific gene and the deletion of an endogenous gene. Efficient transgenic animal production provides numerous opportunities for agriculture and biomedicine. Regulatory agencies around the world have agreed that food derived from cloned animals and their offspring is safe and there is no scientific basis for questioning this. Commercial application of somatic cloning within the EU is via the Novel Food regulation EC No. 258/97. Somatic cloning raises novel questions regarding the ethical and moral status of animals and their welfare which has prompted a controversial discussion in Europe which has not yet been resolved.

The In Vivo Developmental Potential of Porcine Skin-Derived Progenitors and Neural Stem Cells

Multipotent skin-derived progenitors (SKPs) can be traced back to embryonic neural crest cells and are able to differentiate into both neural and mesodermal progeny in vitro. Neural stem cells (NSCs) are capable of self-renewing and can contribute to neuron and glia in the nervous system. Recently, we derived porcine SKPs and NSCs from the same enhanced green fluorescent protein (EGFP) transgenic fetuses and demonstrated that SKPs could contribute to neural and mesodermal lineages in vivo. However, it remains unclear whether porcine SKPs and NSCs can generate ectoderm and mesoderm lineages or other germ layers in vivo. Embryonic chimeras are a well-established tool for investigating cell lineage determination and cell potency through normal embryonic development. Thus, the purpose of this study was to investigate the in vivo developmental potential of porcine SKPs and fetal brain-derived NSCs by chimera production. Porcine SKPs, NSCs, and fibroblasts were injected into precompact in vitro fertilized embryos (IVF) and then transferred into corresponding surrogates 24 h postinjection. We found that porcine SKPs could incorporate into the early embryos and contribute to various somatic tissues of the 3 germ layers in postnatal chimera, and especially have an endodermal potency. However, this developmental potential is compromised when they differentiate into fibroblasts. In addition, porcine NSCs fail to incorporate into host embryos and contribute to chimeric piglets. Therefore, neural crest-derived SKPs may represent a more primitive state than their counterpart neural stem cells in terms of their contributions to multiple cell lineages.

Characterization of the ICSI‐mediated gene transfer method in the production of transgenic pigs

Understanding the behavior of transgenes introduced into oocytes or embryos is essential for evaluating the methodologies for transgenic animal production. We investigated the expression pattern of a transgene transferred to porcine eggs by intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) or pronuclear microinjection (PN injection). The introduction of the EGFP gene by ICSI-MGT yielded significantly more embryos with non-mosaic transgene expression (P < 0.01). In the ICSI-MGT group, 61.5% (24/39) of the embryos were EGFP-positive in all their component blastomeres at the morula stage, while fewer than 10% of such embryos were EGFP-positive in the PN-injection group. Using three types of transgenes, ranging from 3.0 to 7.5 kb in size, we confirmed that approximately one in four fetuses obtained by ICSI-MGT was transgenic, suggesting that ICSI-MGT is a practical method for transgenic pig production. Southern blot analysis of 12 transgenic fetuses produced by ICSI-MGT revealed that the number of integrated transgene copies varied from 1 to 300, with no correlation between transgene size and the number of integrated copies. Fluorescence in situ hybridization analysis revealed that the transgenes were randomly integrated into a single site on the host chromosomes. Together, these data indicate that multiple-copy, single-site integration of a transgene is the primary outcome of ICSI-MGT in the pig and that ICSI-MGT is less likely than PN injection to cause transgene integration in a mosaic manner.

2 THE IN VIVO DEVELOPMENTAL POTENTIAL OF PORCINE SKIN-DERIVED PROGENITORS

Skin-derived progenitors (SKP) are capable of generating both neural and mesodermal progeny in vitro: neurons, Schwann cells, adipocytes, osteocytes and chondrocytes, thus exhibiting characteristics similar to embryonic neural crest stem cells. SKP show distinct transcriptional profiles when compared with neurospheres/neural stem cells in the central nervous system (CNS) and skin-derived fibroblasts, indicating a novel type of multipotent stem cell derived from the dermis of the skin. However, it remains unclear whether SKP cells can produce ectoderm and mesoderm lineages or other germ layers in vivo, although oocyte-like structures can be induced from porcine SKP in vitro. Embryonic chimeras are a well-established tool for investigating cell lineage determination and cell potency through normal embryonic development. Thus the purpose of this study was to investigate the in vivo developmental potential of porcine SKP by chimera production. Porcine SKP cells and fibroblasts were isolated from the back skin of Day 35 to 50 GFP transgenic fetuses. Individual cells or clusters of male GFP transgenic SKP and skin-derived GFP-expressing fibroblasts were injected into pre-compact in vitro-fertilized (IVF) embryos, respectively and then transferred into corresponding surrogates 24 h post-injection. Additional injected embryos were cultured in PZM3 medium for another 2 days until the blastocyst stage and subsequently stained with Hoechst 33342. Interestingly, in some of the chimeras the injected SKP cells migrated and dispersed into different locations of the host blastocysts, whereas in others they remained as a cluster of cells within the chimeric blastocysts. In contrast, the fibroblast cells were not observed to spread around the host blastocysts. Two chimeric fetuses were recovered at the middle of gestation and a litter of viable piglets was born. Genomic DNA was extracted from various tissues of chimeric piglets and subjected to PCR amplification. Two chimeric fetuses and 2 out of 6 piglets carried the GFP transgene in SKP-derived chimeras, but GFP was not present in the fibroblast-derived chimeric fetuses (n = 6). Surprisingly, the GFP transgene was present in various tissues of two SKP-derived chimeric piglets, including lung, heart, liver, artery, kidney, brain, skin, muscle, gut, ovary, pancreas and stomach, thus representing the 3 germ layers (ectoderm, mesoderm and endoderm). In addition, SRY was detected in several tissues of the two GFP-positive female chimeric piglets, confirming the chimerism of these piglets. Therefore, it appears that porcine SKP can contribute to various cell types of the 3 germ layers and have a broader developmental potency than previously expected. Alternatively, pre-compact (4-cell and 8-cell stage) embryos may provide a unique environment for reprogramming skin-derived progenitors into a more primitive state by the process of embryonic compaction. This study was funded by NIH National Center for Research Resources (R01RR013438) and Food for the 21st Century at the University of Missouri.