Replication of somatic micronuclei in bovine enucleated oocytes

Background

Microcell-mediated chromosome transfer (MMCT) was developed to introduce a low number of chromosomes into a host cell. We have designed a novel technique combining part of MMCT with somatic cell nuclear transfer, which consists of injecting a somatic micronucleus into an enucleated oocyte, and inducing its cellular machinery to replicate such micronucleus. It would allow the isolation and manipulation of a single or a low number of somatic chromosomes.

Methods

Micronuclei from adult bovine fibroblasts were produced by incubation in 0.05 μg/ml demecolcine for 46 h followed by 2 mg/ml mitomycin for 2 h. Cells were finally treated with 10 μg/ml cytochalasin B for 1 h. In vitro matured bovine oocytes were mechanically enucleated and intracytoplasmatically injected with one somatic micronucleus, which had been previously exposed [Micronucleus- injected (+)] or not [Micronucleus- injected (?)] to a transgene (50 ng/μl pCX-EGFP) during 5 min. Enucleated oocytes [Enucleated (+)] and parthenogenetic [Parthenogenetic (+)] controls were injected into the cytoplasm with less than 10 pl of PVP containing 50 ng/μl pCX-EGFP. A non-injected parthenogenetic control [Parthenogenetic (?)] was also included. Two hours after injection, oocytes and reconstituted embryos were activated by incubation in 5 μM ionomycin for 4 min + 1.9 mM 6-DMAP for 3 h. Cleavage stage and egfp expression were evaluated. DNA replication was confirmed by DAPI staining. On day 2, Micronucleus- injected (?), Parthenogenetic (?) and in vitro fertilized (IVF) embryos were karyotyped. Differences among treatments were determined by Fisher′s exact test (p≤0.05).

Results

All the experimental groups underwent the first cell divisions. Interestingly, a low number of Micronucleus-injected embryos showed egfp expression. DAPI staining confirmed replication of micronuclei in most of the evaluated embryos. Karyotype analysis revealed that all Micronucleus-injected embryos had fewer than 15 chromosomes per blastomere (from 1 to 13), while none of the IVF and Parthenogenetic controls showed less than 30 chromosomes per spread.

Conclusions

We have developed a new method to replicate somatic micronuclei, by using the replication machinery of the oocyte. This could be a useful tool for making chromosome transfer, which could be previously targeted for transgenesis.

     

The Green Fluorescent Protein Gene Functions as a Reporter of Gene Expression in Phanerochaete chrysosporium

The enhanced green fluorescent protein (GFP) gene (egfp) was used as a reporter of gene expression driven by the glyceraldehyde-p-dehydrogenase (gpd) gene promoter and the manganese peroxidase isozyme 1 (mnp1) gene promoter in Phanerochaete chrysosporium. Four different constructs were prepared. pUGGM3′ and pUGiGM3′ contain the P. chrysosporium gpd promoter fused upstream of the egfp coding region, and pUMGM3′ and pUMiGM3′ contain the P. chrysosporium mnp1 promoter fused upstream of the egfp gene. In all constructs, the egfp gene was followed by the mnp1 gene 3′ untranslated region. In pUGGM3′ and pUMGM3′, the promoters were fused directly with egfp, whereas in pUGiGM3′ and pUMiGM3′, following the promoters, the first exon (6 bp), the first intron (55 bp), and part of the second exon (9 bp) of the gpd gene were inserted at the 5′ end of the egfp gene. All constructs were ligated into a plasmid containing the ura1 gene of Schizophyllum commune as a selectable marker and were used to transform a Ural1 auxotrophic strain of P. chrysosporium to prototrophy. Crude cell extracts were examined for GFP fluorescence, and where appropriate, the extracellular fluid was examined for MnP activity. The transformants containing a construct with an intron 5′ of the egfp gene (pUGiGM3′ and pUMiGM3′) exhibited maximal fluorescence under the appropriate conditions. The transformants containing constructs with no introns exhibited minimal or no fluorescence. Northern (RNA) blots indicated that the insertion of a 5′ intron resulted in more egfp RNA than was found in transformants carrying an intronless egfp. These results suggest that the presence of a 5′ intron affects the expression of the egfp gene in P. chrysosporium. The expression of GFP in the transformants carrying pUMiGM3′ paralled the expression of endogenous mnp with respect to nitrogen and Mn levels, suggesting that this construct will be useful in studying cis-acting elements in the mnp1 gene promoter.     

Natural sesquiterpene lactones induce programmed cell death in Trypanosoma cruzi: A new therapeutic target?

BackgroundChagas disease or American Trypanosomiasis is caused by the flagellated protozoan parasite Trypanosoma cruzi (T. cruzi) and is recognized by the WHO as one of the world's 17 neglected tropical diseases. Only two drugs (Benznidazol, Bz and Nifurtimox, Nx) are currently accepted for treatment, however they cause severe adverse effects and their efficacy is still controversial. It is then important to explore for new drugs.PurposeProgrammed cell death (PCD) in parasites offers interesting new therapeutic targets. The aim of this work was to evaluate the induction of PCD in T. cruzi by two natural sesquiterpene lactones (STLs), dehydroleucodine (DhL) and helenalin (Hln) as compared with the two conventional drugs, Bz and Nx.Material and MethodsHln and DhL were isolated from aerial parts of Gaillardia megapotamica and Artemisia douglassiana Besser, respectively. Purity of compounds (greater than 95%) was confirmed by ¹³C-nuclear magnetic resonance, melting point analysis, and optical rotation. Induction of PCD in T. cruzi epimastigotes and trypomastigotes by DhL, Hln, Bz and Nx was assayed by phosphatidylserine exposure at the parasite surface and by detection of DNA fragmentation using the TUNEL assay. Trypanocidal activity of natural and synthetic compounds was assayed by measuring parasite viability using the MTT method.ResultsThe two natural STLs, DhL and Hln, induce programmed cell death in both, the replicative epimastigote form and the infective trypomastigote form of T. cruzi. Interestingly, the two conventional antichagasic drugs (Bz and Nx) do not induce programmed cell death. A combination of DhL and either Bz or Nx showed an increased effect of natural compounds and synthetic drugs on the decrease of parasite viability.ConclusionDhL and Hln induce programmed cell death in T. cruzi replicative epimastigote and infective trypomastigote forms, which is a different mechanism of action than the conventional drugs to kill the parasite. Therefore DhL and Hln may offer an interesting option for the treatment of Chagas disease, alone or in combination with conventional drugs.     

Novel methods to induce exogenous gene expression in SCNT, parthenogenic and IVF preimplantation bovine embryos

The import of exogenous DNA (eDNA) from the cytoplasm to the nucleus represents a key intracellular obstacle for efficient gene delivery in mammalian cells. In this study, cumulus cells or oolemma vesicles previously incubated with eDNA, and naked eDNA were injected into the cytoplasm of MII oocytes to evaluate their efficiency for eDNA expressing bovine embryo production. Our study evaluated the potential of short time co-incubation (5 min) of eDNA with; (1) cumulus cells, to be used as donor cells for SCNT and (2) oolemma vesicles (vesicles) to produce parthenogenic transgene expressing embryos. In addition, we included a group consisting of the injection of eDNA alone (plasmid) followed by parthenogenic activation. Two different pCX-EGFP plasmid concentrations (50 and 500 ng/μl) were employed. The results showed that embryos produced by SCNT and by vesicle injection assisted by chemical activation were able to express the eDNA in higher rates than embryos injected with plasmid alone. The lower plasmid concentration allowed the highest development rates in all groups. Using confocal microscopy, we analyzed the interaction of FITC- labeled eDNA with cumulus cells and vesicles as well as oocytes injected with labeled plasmid alone. Our images demonstrated that eDNA interacted with cumulus cells and vesicles, resulting an increase in its expression efficiency. In contrast, oocytes injected with DNA alone did not show signs of transgene accumulation, and their eDNA expression rates were lower. In a further experiment, we evaluated if transgene-expressing embryos could be produced by means of vesicle injection followed by IVF. The lower plasmid concentration (50 ng/μl) injected after IVF, produced the best results. Preliminary FISH analysis indicated detectable integration events in 1/5 of SCNT blastocysts treated. Our studies demonstrate for the first time that short term transgene co-incubation with somatic cells can produce transgene-expressing mammalian SCNT embryos and also that parthenogenic, eDNA- expressing embryos can be obtained by injection of vesicles or eDNA alone. Moreover, eDNA-expressing embryos can be also obtained by cytoplasmic injection vesicles in IVF zygotes, simplifying the traditional IVF pronuclear injection technique.     

ACTH Induces Cav3.2 Current and mRNA by cAMP-dependent and cAMP-independent Mechanisms

Bovine adrenal zona fasciculata (AZF) cells express Ca_v3.2 T-type Ca²⁺ channels that function pivotally in adrenocorticotropic hormone (ACTH)-stimulated cortisol secretion. The regulation of Ca_v3.2 expression in AZF cells by ACTH, cAMP analogs, and their metabolites was studied using Northern blot and patch clamp recording. Exposing AZF cells to ACTH for 3–6 days markedly enhanced the expression of Ca_v3.2 current. The increase in Ca_v3.2 current was preceded by an increase in corresponding CACNA1H mRNA. O-Nitrophenyl,sulfenyl-adrenocorticotropin, which produces a minimal increase in cAMP, also enhanced Ca_v3.2 current. cAMP analogs, including 8-bromoadenosine cAMP (600 μm) and 6-benzoyladenosine cAMP (300 μm) induced CACNA1H mRNA, but not Ca_v3.2 current. In contrast, 8-(4-chlorophenylthio) (8CPT)-cAMP (10–50 μm) enhanced CACNA1H mRNA and Ca_v3.2 current, whereas nonhydrolyzable Sp-8CPT-cAMP failed to increase either Ca_v3.2 current or mRNA. Metabolites of 8CPT-cAMP, including 8CPT-adenosine and 8CPT-adenine, increased Ca_v3.2 current and mRNA with a potency and effectiveness similar to the parent compound. The Epac activator 8CPT-2′-O-methyl-cAMP and its metabolites 8CPT-2′-OMe-5′-AMP and 8CPT-2′-O-methyl-adenosine increased CACNA1H mRNA and Ca_v3.2 current; Sp-8CPT-2′-O-methyl-cAMP increased neither Ca_v3.2 current nor mRNA. These results reveal an interesting dichotomy between ACTH and cAMP with regard to regulation of CACNA1H mRNA and Ca²⁺ current. Specifically, ACTH induces expression of CACNA1H mRNA and Ca_v3.2 current in AZF cells by mechanisms that depend at most only partly on cAMP. In contrast, cAMP enhances expression of CACNA1H mRNA but not the corresponding Ca²⁺ current. Surprisingly, chlorophenylthio-cAMP analogs stimulate the expression of Ca_v3.2 current indirectly through metabolites. ACTH and the metabolites may induce Ca_v3.2 expression by the same, unidentified mechanism.     

Calcium moderation of cadmium stress explored using a stress-inducible transgenic strain of Caenorhabditis elegans

In a transgenic strain of Caenorhabditis elegans carrying a stress-inducible lacZ reporter gene, short-term sublethal exposure to heavy metals activates transgene expression. The transgene response to Cd²⁺ is strongly inhibited by Ca²⁺ ions; furthermore, Ca²⁺ reduces the net accumulation of Cd²⁺ by worms. Both Ca²⁺ and a variety of calcium uptake inhibitors (nifedipine, La³⁺, verapamil) depress the dose response of the transgene to Cd²⁺. Calcium ionophore (A23187) slightly increases transgene activity in control and Cd²⁺ treated worms, but has a much larger effect in the case of Mn²⁺, reflecting its much greater affinity for this ion.     

Permeation of a β-heptapeptide derivative across phospholipid bilayers

Based on a number of experiments it is concluded that the fluorescein labeled β-heptapeptide fluoresceinyl-NH-CS-(S)-β³hAla-(S)-β³hArg-(R)-β³hLeu-(S)-β³hPhe-(S)-β³hAla-(S)-β³hAla-(S)-β³hLys-OH translocates across lipid vesicle bilayers formed from DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine). The conclusion is based on the following observations: (i) addition of the peptide to the vicinity of micrometer-sized giant vesicles leads to an accumulation of the peptide inside the vesicles; (ii) if the peptide is injected inside individual giant vesicles, it is released from the vesicles in a time dependent manner; (iii) if the peptide is encapsulated within sub-micrometer-sized large unilamellar vesicles, it is released from the vesicles as a function of time; (iv) if the peptide is submitted to immobilized liposome chromatography, the peptide is retained by the immobilized DOPC vesicles. Furthermore, the addition of the peptide to calcein-containing DOPC vesicles does not lead to significant calcein leakage and vesicle fusion is not observed. The finding that derivatives of the β-heptapeptide (S)-β³hAla-(S)-β³hArg-(R)-β³hLeu-(S)-β³hPhe-(S)-β³hAla-(S)-β³hAla-(S)-β³hLys-OH can translocate across phospholipid bilayers is supported by independent measurements using Tb³⁺-containing large unilamellar vesicles prepared from egg phosphatidylcholine and wheat germ phosphatidylinositol (molar ratio of 9:1) and a corresponding peptide that is labeled with dipicolinic acid instead of fluorescein. The experiments show that this dipicolinic acid labeled β-heptapeptide derivative also permeates across phospholipid bilayers. The possible mechanism of the translocation of the particular β-heptapeptide derivatives across the membrane of phospholipid vesicles is discussed within the frame of the current understanding of the permeation of certain oligopeptides across simple phospholipid bilayers.     

Efficient somatic embryogenesis and Agrobacterium-mediated transformation of pothos (Epipremnum aureum) ‘Jade’

Leaf and petiole explants of monocotyledonous pothos (Epipremnum aureum) ‘Jade’ were cultured on Murashige and Skoog basal medium supplemented with N-(2-chloro-4-pyridl)-N′-phenylurea (CPPU) or N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (TDZ) with α-naphthalene acetic acid (NAA). Somatic embryos appeared directly from explants after 4–8 weeks of culture; 9.1 μM TDZ with 1.1 μM NAA induced 61.1 % leaf discs and 94.4 % of petiole segments to produce plantlets through embryo conversion. Using this established regeneration method and an enhanced green fluorescent protein (GFP) gene (egfp) as a reporter marker, an Agrobacterium-mediated transformation procedure was developed. Leaf discs and petiole segments were inoculated with Agrobacterium tumefaciens strain EHA105 harboring a binary vector pLC902 that contains novel bi-directional duplex promoters driving the egfp gene and hygromycin phosphotransferase gene (hpt), respectively. The explants were co-cultivated with strain EHA105 for 3, 5, and 7 days, respectively prior to selective culture with 25 mg l^?1 hygromycin. A 5-day co-cultivation led to 100 % of leaf discs to show transient GFP expression and 23.8 % of the discs to produce stable GFP-expressing somatic embryos. A 7-day co-cultivation of petiole explants resulted in the corresponding responses at 100 and 14.3 %, respectively. A total of 237 transgenic plants were obtained, and GFP fluorescence was observed in all plant organs. Regular PCR and quantitative real-time PCR analyses confirmed the presence of 1 or 2 copies of the egfp gene in analyzed plants. The highly efficient regeneration and transformation systems established in this study may enable genetic improvement of this vegetatively propagated species through biotechnological means.     

Effect of dietary fish oil on selected inflammatory markers in pigs

The present study tested a hypothesis that dietary fish oil (eicosapentaenoic acid+docosahexaenoic acid) in a commonly achievable dose ameliorates a systemic inflammation in pigs. Two groups of pigs of 16 animals each were fed a diet with either 2.5% of fish oil (F) or a control diet with 2.5% of palm oil (P). After 70 days of fattening, eight F and eight P pigs were challenged (F⁺; P⁺) i.v. by lipopolysaccharide. After 3 h, all pigs were sacrificed and blood, liver and visceral adipose tissue (VAT) samples were taken. No significant effect (P>0.05) of dietary oil on the feed intake and daily weight gain was found out. Less neutrophils (16.8% v. 28.8%; P<0.05) were found in the F⁺-leukocytes of the peripheral blood; F⁺ pigs had lower (P<0.05) percentage of the swine leukocyte antigen-D-related CD163⁺ (SLA-DR⁺ CD163⁺) macrophages in the VAT (15.4% v. 21.8%) and lower expression of the SLA-DR-CD163⁺ surface molecules of the VAT macrophages. No difference (P>0.05) between F⁺ and P⁺ pigs in the peroxisome proliferator-activated receptor γ, GPR120, Adipor1 and Adipor2 (adiponectin receptor) gene expression, respectively, was established; plasma adiponectin was the same (21.1 ng/ml) in F⁺ and P⁺ pigs. In comparison with the P⁺ pigs, increased expression of the lipopolysaccharide-binding protein (LBP) gene and intercellular adhesion molecule 1 (ICAM1) gene was found out in the liver of the F⁺ pigs; expression of the tumor necrosis factor α (TNFα) gene was higher in the liver but lower in the VAT of the F⁺ pigs (P<0.05). The F⁺ pigs had higher (P<0.05) plasma concentration of both anti-inflammatory cytokine interleukin-4 (0.46 v. 0.04 ng/ml) and pro-inflammatory TNF-α (13.41 v. 7.72 ng/ml). It was concluded that dietary fish oil at the tested amount had a negligible effect on expression of the evaluated receptor genes and plasma adiponectin, and had an ambiguous effect on expression of cytokine genes and plasma cytokine levels.     

C-Terminal Modulatory Domain Controls Coupling of Voltage-Sensing to Pore Opening in Cav1.3 L-type Ca Channels

Activity of voltage-gated Ca_v1.3 L-type Ca²⁺ channels is required for proper hearing as well as sinoatrial node and brain function. This critically depends on their negative activation voltage range, which is further fine-tuned by alternative splicing. Shorter variants miss a C-terminal regulatory domain (CTM), which allows them to activate at even more negative potentials than C-terminally long-splice variants. It is at present unclear whether this is due to an increased voltage sensitivity of the Ca_v1.3 voltage-sensing domain, or an enhanced coupling of voltage-sensor conformational changes to the subsequent opening of the activation gate. We studied the voltage-dependence of voltage-sensor charge movement (Q_ON-V) and of current activation (I_Ca-V) of the long (Ca_v1.3_L) and a short Ca_v1.3 splice variant (Ca_v1.3_42A) expressed in tsA-201 cells using whole cell patch-clamp. Charge movement (Q_ON) of Ca_v1.3_L displayed a much steeper voltage-dependence and a more negative half-maximal activation voltage than Ca_v1.2 and Ca_v3.1. However, a significantly higher fraction of the total charge had to move for activation of Ca_v1.3 half-maximal conductance (Ca_v1.3: 68%; Ca_v1.2: 52%; Ca_v3.1: 22%). This indicated a weaker coupling of Ca_v1.3 voltage-sensor charge movement to pore opening. However, the coupling efficiency was strengthened in the absence of the CTM in Ca_v1.3_42A, thereby shifting I_Ca-V by 7.2 mV to potentials that were more negative without changing Q_ON-V. We independently show that the presence of intracellular organic cations (such as n-methyl-D-glucamine) induces a pronounced negative shift of Q_ON-V and a more negative activation of I_Ca-V of all three channels. These findings illustrate that the voltage sensors of Ca_v1.3 channels respond more sensitively to depolarization than those of Ca_v1.2 or Ca_v3.1. Weak coupling of voltage sensing to pore opening is enhanced in the absence of the CTM, allowing short Ca_v1.3_42A splice variants to activate at lower voltages without affecting Q_ON-V.     

C-Terminal Modulatory Domain Controls Coupling of Voltage-Sensing to?Pore Opening in Cav1.3 L-type Ca2+ Channels

Activity of voltage-gated Ca_v1.3 L-type Ca²⁺ channels is required for proper hearing as well as sinoatrial node and brain function. This critically depends on their negative activation voltage range, which is further fine-tuned by alternative splicing. Shorter variants miss a C-terminal regulatory domain (CTM), which allows them to activate at even more negative potentials than C-terminally long-splice variants. It is at present unclear whether this is due to an increased voltage sensitivity of the Ca_v1.3 voltage-sensing domain, or an enhanced coupling of voltage-sensor conformational changes to the subsequent opening of the activation gate. We studied the voltage-dependence of voltage-sensor charge movement (Q_ON-V) and of current activation (I_Ca-V) of the long (Ca_v1.3_L) and a short Ca_v1.3 splice variant (Ca_v1.3_42A) expressed in tsA-201 cells using whole cell patch-clamp. Charge movement (Q_ON) of Ca_v1.3_L displayed a much steeper voltage-dependence and a more negative half-maximal activation voltage than Ca_v1.2 and Ca_v3.1. However, a significantly higher fraction of the total charge had to move for activation of Ca_v1.3 half-maximal conductance (Ca_v1.3: 68%; Ca_v1.2: 52%; Ca_v3.1: 22%). This indicated a weaker coupling of Ca_v1.3 voltage-sensor charge movement to pore opening. However, the coupling efficiency was strengthened in the absence of the CTM in Ca_v1.3_42A, thereby shifting I_Ca-V by 7.2 mV to potentials that were more negative without changing Q_ON-V. We independently show that the presence of intracellular organic cations (such as n-methyl-D-glucamine) induces a pronounced negative shift of Q_ON-V and a more negative activation of I_Ca-V of all three channels. These findings illustrate that the voltage sensors of Ca_v1.3 channels respond more sensitively to depolarization than those of Ca_v1.2 or Ca_v3.1. Weak coupling of voltage sensing to pore opening is enhanced in the absence of the CTM, allowing short Ca_v1.3_42A splice variants to activate at lower voltages without affecting Q_ON-V.     

KIT is required for hepatic function during mouse post-natal development

Background

Targeted disruption of the murine 3β-hydroxysterol-Δ7-reductase gene (Dhcr7), an animal model of Smith-Lemli-Opitz syndrome, leads to loss of cholesterol synthesis and neonatal death that can be partially rescued by transgenic replacement of DHCR7 expression in brain during embryogenesis. To gain further insight into the role of non-brain tissue cholesterol deficiency in the pathophysiology, we tested whether the lethal phenotype could be abrogated by selective transgenic complementation with DHCR7 expression in the liver.

Results

We generated mice that carried a liver-specific human DHCR7 transgene whose expression was driven by the human apolipoprotein E (ApoE) promoter and its associated liver-specific enhancer. These mice were then crossed with Dhcr7+/- mutants to generate Dhcr7-/- mice bearing a human DHCR7 transgene. Robust hepatic transgene expression resulted in significant improvement of cholesterol homeostasis with cholesterol concentrations increasing to 80~90 % of normal levels in liver and lung. Significantly, cholesterol deficiency in brain was not altered. Although late gestational lung sacculation defect reported previously was significantly improved, there was no parallel increase in postnatal survival in the transgenic mutant mice.

Conclusion

The reconstitution of DHCR7 function selectively in liver induced a significant improvement of cholesterol homeostasis in non-brain tissues, but failed to rescue the neonatal lethality of Dhcr7 null mice. These results provided further evidence that CNS defects caused by Dhcr7 null likely play a major role in the lethal pathogenesis of Dhcr7 ^-/- mice, with the peripheral organs contributing the morbidity.     

Highly efficient transfer and stable expression of two genes upon lentivirus transduction of mesenchymal stem cells from human bone marrow

The efficiency of human bone marrow (BM) mesenchymal stem cell (MSC) transduction with a bicistronic lentivirus vector was estimated, and the stability of transgene expression in genetically modified MSCs was determined. First-passage BM MSCs were capable of efficient transduction with the bicistronic lentivirus vector. The transduction efficiency depended on the multiplicity of infection (MOI), being 64.64 ± 6.5 and 88.6 ± 2.9% at MOI 10 and 20, respectively. The lentivirus transduction efficiency proved independent on the number of passages of a BM MSC culture, and expression of the egfp and dsRed1 transgenes in genetically modified MSCs remained stable for one month of culturing. A comparison showed that the level of egfp and dsRed1 transgene expression was preserved upon hepatogenic differentiation in vitro. The results provide a basis for further development of multigenic modification of human BM MSCs for research and/or therapeutic purposes.     

Chloroplast-targeted expression of the codon-optimized truncated cry1Ah gene in transgenic tobacco confers a high level of protection against insects

Key message

The study of insect-resistant transgenic tobacco provides a good foundation for the further application of the cry1Ah gene in other important crops.

Abstract

To improve transgene expression levels and insect resistance, the coding sequence of the novel Bacillus thuringiensis insecticidal gene cry1Ah (truncated cry1Ah) was modified according to the codon bias of the plant by increasing its GC content from the original 37 % to 48, 55, and 63 % (designated m1-cry1Ah, m2-cry1Ah, and m3-cry1Ah, respectively). In addition, the m3-cry1Ah gene was linked with a transit peptide sequence for chloroplast-targeted expression (designated ctp-m3-cry1Ah). Four plant expression vectors were constructed harboring m1-cry1Ah, m2-cry1Ah, m3-cry1Ah, or ctp-m3-cry1Ah. A total of 23 transgenic tobacco lines were produced with the four constructs by Agrobacterium tumefaciens-mediated transformation. PCR, Southern hybridization, quantitative RT-PCR and ELISA indicated that the cry1Ah gene was not only integrated into the tobacco genome, but was also successfully expressed at the mRNA and protein levels. The Cry1Ah protein level in ctp-m3-cry1Ah plants reached 4.42 μg/g fresh weight, which was a 2- to 10-fold increase over the levels observed in m1-cry1Ah, m2-cry1Ah, and m3-cry1Ah plants and resulted in the highest resistance to Helicoverpa armigera based on bioassays. Our results demonstrated that combining the codon optimization of cry1Ah gene with the targeting of Cry1Ah protein to the chloroplasts conferred a high level of protection against insects. The results of our experiments in tobacco, an important model system, provide a good foundation for enhancing the insecticidal efficacy of staple crops.     

Production and Characterization of Novel Recombinant Adeno-Associated Virus Replicative-Form Genomes: A Eukaryotic Source of DNA for Gene Transfer

Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms of a eukaryotic vector DNA that consist solely of an expression cassette flanked by adeno-associated virus (AAV) inverted terminal repeats. Extensive structural analyses revealed that this AAV-derived vector DNA consists of linear, duplex molecules with covalently closed ends (termed closed-ended, linear duplex, or “CELiD”, DNA). CELiD vectors, produced in Sf9 insect cells, require AAV rep gene expression for amplification. Amounts of CELiD DNA produced from insect cell lines stably transfected with an ITR-flanked transgene exceeded 60 mg per 5×10⁹ Sf9 cells, and 1–15 mg from a comparable number of parental Sf9 cells in which the transgene was introduced via recombinant baculovirus infection. In mice, systemically delivered CELiD DNA resulted in long-term, stable transgene expression in the liver. CELiD vectors represent a novel eukaryotic alternative to bacterial plasmid DNA.     

Effects of dietary calcium and phosphorus deficiency and subsequent recovery on broiler chicken growth performance and bone characteristics

The ability of birds to modify dietary phosphorus utilisation when fed with low-phosphorus and calcium (Ca) diets was studied using different sequences of dietary phosphorus and Ca restriction (depletion) and recovery (repletion) during the grower and the finisher phases. A total of 3600 Ross 708 broilers were randomly divided into 10 replicate pens per treatment (60 per pen, six pens per block). Chicks were fed a common starter diet from days 0 to 10, then a grower control diet (C: 0.90% Ca, 0.39% non-phytate phosphorus, nPP), mid-level diet (M: 0.71% Ca, 0.35% nPP) or low Ca and nPP diet (L: 0.60% Ca, 0.30% nPP) from days 11 to 21, followed by a finisher diet C, M or L containing, respectively, 0.85%, 0.57% or 0.48% Ca and 0.35%, 0.29% or 0.24% nPP from days 22 to 37. Six treatment sequences were tested: CC, MM, LL, ML, LC and LM. Bone mineral content by dual-energy X-ray, tibia ash, toe ash weight and tibia breaking strength were measured on days 21 and 37. No significant effect was observed on growth performance throughout the experiment. Diet L reduced bone mineral content, breaking strength, tibia and toe ash by 9%, 13%, 11% and 10%, respectively, on day 21 (compared with diet C, for linear effect, P<0.05). On day 37, bone mineral content, breaking strength, tibia and toe ash remained lower compared with control values (CC v. MM v. LL, P<0.05 for linear and quadratic effects). Mineral depletion duration (ML v. LL) did not affect bone mineral status. Replenishing with the C diet during the finisher phase (LC) restored bone mineral content, tibia ash and toe ash weight better than the M diet did, but not to control levels (CC v. LC v. LM, for linear effect, P<0.05). These results confirm that dietary Ca and nPP may be reduced in the grower phase without affecting final growth performance or breaking strength as long as the finisher diet contains sufficient Ca and nPP. The practical applications of this strategy require further study in order to optimise the depletion and repletion steps.     

Transfer of maternally administered fusogenic liposome-DNA complexes into monkey fetuses in a pregnancy model

Background

Materno‐fetal transfer of intravenously administered liposome‐plasmid DNA complexes has been demonstrated only in mice. Studies on its materno‐fetal transfer in the pregnant monkey model is needed because of critical differences in placental structure between primates including humans and rodents.

Methods

The reporter plasmid pEGFP‐C1 was formulated in cationic lipid containing polybrene and vesicular stomatitis virus G protein. The fusogenic liposome‐plasmid DNA complexes were intradermally injected into pregnant common marmosets (N=2), a New World monkey, near term. DNA extracted from fetal tissues was subjected to PCR for detection of the egfp gene. Confocal microscopy and immunostaining were performed to determine the sites of transgene expression in the fetal organs.

Results

The egfp gene was detected in fetal blood and major organs (heart, liver, lung). The encoded protein was mainly produced in the endothelial cells of blood vessels in the fetal lungs.

Conclusions

This is the first report on materno‐fetal transfer of intradermally administered fusogenic liposome‐plasmid DNA complexes and fetal expression of a transgene in primates. Copyright © 2002 John Wiley & Sons, Ltd.

     

Unstable expression of transgene is associated with the methylation of CAG promoter in the offspring from the same litter of homozygous transgenic mice

Transgenic animals have been established for studying gene function, improving animals’ production traits, and providing organ models for the exploration of human diseases. However, the stability of inheritance and transgene expression in transgenic animals has gained extensive attention. The unstable expression of transgene through DNA methyltransferase (DNMT) targeting to the methylation of transgenic DNA such as CAG promoter and Egfp coding region in homozygous transgenic animals is still unknown. In the present study, the offspring from the same litter of homozygous transgenic mice carrying ubiquitously expressed enhanced green fluorescence protein driven by CMV early enhancer/chicken β-actin (CAG) promoter was observed to have unstable expression of transgene Egfp, quantitative PCR, western blot and bisulfite sequencing were conducted to quantify the expressional characteristics and methylation levels in various tissues. The correlation between transgene expression and methylation was analyzed. We have found that transgene expression is dependent on the methylation of CAG promoter, but not Egfp coding region. We have also characterized the correlation between the methylation of CAG promoter and DNMT, and found that only Dnmt3b expression is correlated with the methylation of CAG promoter. In conclusion, Dnmt3b-related methylation of CAG promoter can inhibit the transgene expression and may result in the unstable expression of transgene in the offspring from the same litter of homozygous transgenic mice.