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.

First Report of a Huanglongbing-Like Disease of Citrus in Sao Paulo State, Brazil and Association of a New Liberibacter Species, "Candidatus Liberibacter americanus", with the Disease.

Huanglongbing (HLB) (ex-greening) is one of the most serious diseases of citrus. The causal agent is a noncultured, sieve tube-restricted α-proteobacterium, "Candidatus Liberibacter africanus" in Africa and "Candidatus Liberibacter asiaticus" in Asia (2). The disease has never been reported from the American continent. However, Diaphorina citri, the Asian psyllid vector of HLB, is found in South, Central, and North America (Florida and Texas). Early in 2004, leaf and fruit symptoms resembling those of HLB were observed in several sweet orange orchards near the city of Araraquara, Sao Paulo State. Leaf mottling on small and large leaves was the major symptom. Shoots with affected leaves were yellowish. Fruits were small and lopsided, contained many aborted seeds, and appeared more severely affected than were plants infected with classic HLB. Forty-three symptomatic samples and twenty-five samples of symptomless sweet orange leaves from five farms were analyzed for the presence of the HLB-liberibacters using polymerase chain reaction (PCR) with two sets of HLB-specific primers for amplification of 16S rDNA (2,3) and ribosomal protein genes (1). None of the 43 symptomatic leaf samples gave a positive PCR amplification, while HLB-affected leaves from the Bordeaux HLB collection produced the characteristic amplicons with both sets of primers. The 43 symptomatic and the 25 symptomless leaf samples were then analyzed using PCR with universal primers for amplification of bacterial 16S rDNA (4). All symptomatic leaf samples, but none of the symptomless leaf samples, yielded the same 16S rDNA amplification product, indicating the presence of a bacterium in the symptomatic leaves. This was confirmed using the observation of a sieve tube restricted bacterium by electron microscopy. The 16S rDNA product was cloned, sequenced, and compared with those of "Ca. L. africanus" and "Ca. L. asiaticus". While the 16S rDNAs of these two liberibacter species have 97.5% sequence identity, the 16S rDNA sequence of the new bacterium shared only 93.7% identity with that of "Ca. L. asiaticus" and 93.9% with that of "Ca. L. africanus". The 16S rDNA sequence of the new bacterium had a secondary loop structure characteristic of the α subdivision of the proteobacteria and possessed all the oligonucleotide signatures characteristic of the liberibacters. For these reasons, the new bacterium is a liberibacter and is sufficiently different phylogenetically from known liberibacters to warrant a new species, "Candidatus Liberibacter americanus". Specific PCR primers for amplification of the 16S rDNA of the new species have been developed. They were able to detect "Ca. L. americanus" in 214 symptomatic leaf samples from 47 citrus farms in 35 municipalities, while the "old" species, "Ca. L. asiaticus", has been found only four times within the 47 farms. References: (1) A. Hocquellet et al. Mol. Cell. Probes, 13:373, 1999. (2) S. Jagoueix et al. Int. J. Syst. Bacteriol. 44:379, 1994. (3) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (4) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

Analysis and quantitation of rotenoids and flavonoids in Derris (Lonchocarpus urucu) by high-temperature high-resolution gas chromatography.

A glass capillary column coated with PS-086 (15% phenyl-80% methylpolysiloxane, 15 m x 0.30-mm i.d., 0.1-microm film thickness) is used to analyze extracts from Lonchocarpus urucu (Derris urucu). Several secondary metabolites (8 flavonoids, 10 rotenoids) are characterized without derivatization, and the rotenoids are quantitated by high-temperature high-resolution gas chromatography (HTHRGC) and HTHRGC coupled with mass spectrometry (HTHRGC-MS). The limit of detection in flame ionization detection of rotenone is approximately 0.5 microg/mL, and the limit of quantitation was 2 microg/mL. Derris urucu bark is an excellent source of rotenone isomers (80 mg/g), deguelin (30 mg/g), and rotenolone (26 mg/g). Single solvent extractions (hexane, methylene dichloride, acetone, or methanol) are not able to fully extract the flavonoids and rotenoids. Complete extraction is achieved using a mixture of methanol-methylene dichloride (1:1), indicating a complex association of these compounds with the plant tissue. HTHRGC and HTHRGC-MS are shown to be quick and informative tools for the rapid analysis of crude extracts without the need for prior derivatization and fractionation.