Transfection and expression of exogenous gene in laying hens oviduct in vitro and in vivo

To examine whether or not the regulatory sequence of chicken ovalbumin gene can drive transgene expression specifically in hen oviduct, the authors constructed an oviduct-specific expression vector (pOV), containing 3.0 kilobases (kb) of the 5'-flanking sequence and 3.0 kb of the 3'-flanking sequence of the chicken ovalbumin gene. Jellyfish green fluorescence protein (EGFP) reporter gene and bacterial LacZ reporter gene were respectively inserted into the downstream of the 5'-regulatory region. The recombinants were named as pOVEGFP and pOVLacZ. Two transfer systems, in vitro and in vivo, were used to verify the function of the vector. In vitro, the plasmid DNA pOVEGFP and pEGFP-N1 were transfected respectively by the polyethyle-neimine procedure into the primary chicken oviduct epithelium (PCOE) and fibroblasts cells isolated from laying hens. In vivo, the recombinant vector pOVLacZ was injected into egg-laying hens via wing vein and the tissues were collected for RT-PCR analysis. The results showed that expression of pEGFP-Nl was achieved at low level in oviduct epithelial cells and at high level in fibroblasts, but that the recombinant vector was not expressed in both cells. RT-PCR analysis showed that the LacZ gene was transcribed in the oviduct, but not in the heart, liver, kidney and spleen of the injected hens. Accordingly, the β-galactosidase activity was only detected in the oviduct magnum (116.7 mU/ml) and eggs (16.47 mU/ml). These results indicated that the cloned regulation regions of chicken ovalbumin gene could drive exogenous gene expression specifically in the oviducts of hens. In vivo gene injection via wing vein may serve as a rapid production system of recombinant proteins in chicken eggs. In addition, the cultured primary oviduct cells from laying hens were not efficient temporary expression systems for analyzing the function of regulating elements of ovalbumin gene.

Screening Helicobacter pylori genes induced during infection of mouse stomachs

AIM:To investigate the effect of in vivo environment on gene expression in Helicobacter pylori(H.pylori) as it relates to its survival in the host.METHODS:In vivo expression technology(IVET) systems are used to identify microbial virulence genes.We modified the IVET-transcriptional fusion vector,pIVET8,which uses antibiotic resistance as the basis for selection of candidate genes in host tissues to develop two unique IVET-promoter-screening vectors,pIVET11 and pIVET12.Our novel IVET systems were developed by the fusion of random Sau3A DNA fragments of H.pylori and a tandem-reporter system of chloramphenicol acetyltransferase and beta-galactosidase.Additionally,each vector contains a kanamycin resistance gene.We used a mouse macrophage cell line,RAW 264.7 and mice,as selective media to identify specific genes that H.pylori expresses in vivo.Gene expression studies were conducted by infecting RAW 264.7 cells with H.pylori.This was followed by real time polymerase chain reaction(PCR) analysis to determine the relative expression levels of in vivo induced genes.RESULTS:In this study,we have identified 31 in vivo induced(ivi) genes in the initial screens.These 31 genes belong to several functional gene families,including several well-known virulence factors that are expressed by the bacterium in infected mouse stomachs.Virulence factors,vacA and cagA,were found in this screen and are known to play important roles in H.pylori infection,colonization and pathogenesis.Their detection validates the efficacy of these screening systems.Some of the identified ivi genes have already been implicated to play an important role in the pathogenesis of H.pylori and other bacterial pathogens such as Escherichia coli and Vibrio cholerae.Transcription profiles of allivi genes were confirmed by real time PCR analysis of H.pylori RNA isolated from H.pylori infected RAW 264.7 macrophages.We compared the expression profile of H.pylori and RAW 264.7 coculture with that of H.pylori only.Some genes such as cag A,vac A,lpx C,mur I,tlp C,trx B,sod B,tnp B,pgi,rbf A and inf B showed a 2-20 fold upregulation.Statistically significant upregulation was obtained for all the above mentioned genes(P < 0.05).tlp C,cag A,vac A,sod B,rbf A,inf B,tnp B,lpx C and mur I were also significantly upregulated(P < 0.01).These data suggest a strong correlation between results obtained in vitro in the macrophage cell line and in the intact animal.CONCLUSION:The positive identification of these genes demonstrates that our IVET systems are powerful tools for studying H.pylori gene expression in the host environment.

Molecular identification and enzymatic properties of laccase2 from the diamondback moth Plutella xylostella (Lepidoptera:Plutellidae)

Laccase（EC 1.10.3.2）is known to oxidize various aromatic and nonaromatic compounds via a radical-catalyzed reaction,which generally includes two types of laccase,Lac1 and Lac2.Lac1 oxidizes toxic compounds in the diet,and Lac2 is known to play an important role in melanizing the insect exoskeleton.In this study,we cloned and sequenced the cDNA of the diamondback moth,Plutella xylostella Lac2（PxLac2）,from the third instar larvae using polymerase chain reaction（PCR）and rapid amplification of cDNA ends techniques.The results showed that the full-length PxLac2 cDNA was 1 944 bp long and had an open reading frame of 1 794 bp.PxLac2 encoded a protein with 597 amino acids and had a molecular weight of 66.09 kDa.Moreover,we determined the expression levels of PxLac2 in different stages by quantitative PCR（qPCR）.The results indicated that PxLac2 was expressed differently in different stages.We observed the highest expression level in pupae and the lowest expression level in fourth instar larvae.We also investigated the enzymatic properties of laccase,which had optimal activity at pH 3.0 and at 35°C.Under these optimal conditions,laccase had a Michaelis constant（Km）of 0.97 mmol L-1,maximal reaction speed（Vm）of 56.82 U mL-1,and activation energy（Ea）of 17.36 kJ mol-1 to oxidize2,2＇-azino-bis（3-ethylbenzothiazoline-6-sulfonic acid ammonium salt）.Type II copper enhanced laccase activity below 0.8mmol L-1 and reduced enzyme activity above 0.8 mmol L-1 with an IC50 concentration of 1.26 mmol L-1.This study provides insights into the biological function of laccase.

Screening and identification of Shigella flexneri 2a virulence-related genes induced after invasion of epithelial cells

An in vivo expression technology (IVET) was applied to screen s.flexneri 2a genes induced after invasion of epithelial cells, and virulence-related genes were further identified by mutational analysis. Thirteen intracellular induced genes were identified with a HeLa cell infection model. Of these, two were identified as alkylation-related genes; one was related to metabolism; one encoded a transcriptional regulator; three were identified as insertion elements; three ap- peared to be antisense to genes involved in the transmethylation,biosyntheseis, and phos- photransferase system;and three were predicted to encode polypeptides with unknown functions. Intracellular survival assavs showed that the mutants of alkA,citC and wcaJ genes had lower capability of intracellular replication or survival than the the wild-type strain.The results indicated that alkA, citC and wcaJ genes could take part in the intracellular survival or replication of S. flexneri 2a and the capability of intracellular survival or replication could be one of the major virulence elements. However, the yaiC mutant was able to survive in the murine infection assay but almost not in HeLa cell infection assay. Very possibly, yaiC gene was involved in the other mechanism of S. flexneri virulence. This study might lead to a better understanding of the intra- cellular survival or proliferation process of S. flexneri 2a and perhaps provide insights into the pathogenicity of this pathogen.