Development and characterization of a guinea pig model for Marburg virus.

The Angolan strain of Marburg virus (MARV/Ang) can cause lethal disease in humans with a case fatality rate of up to 90%, but infection of immunocompetent rodents do not result in any observable symptoms. Our previous work includes the development and characterization of a MARV/Ang variant that can cause lethal disease in mice (MARV/Ang-MA), with the aim of using this tool to screen for promising prophylactic and therapeutic candidates. An intermediate animal model is needed to confirm any findings from mice studies before testing in the gold-standard non-human primate (NHP) model. In this study, we serially passaged the clinical isolate of MARV/Ang in the livers and spleens of guinea pigs until a variant emerged that causes 100% lethality in guinea pigs (MARV/Ang-GA). Animals infected with MARV/Ang-GA showed signs of filovirus infection including lymphocytopenia, thrombocytopenia, and high viremia leading to spread to major organs, including the liver, spleen, lungs, and kidneys. The MARV/Ang-GA guinea pigs died between 7-9 days after infection, and the LD50 was calculated to be 1.1×10-1 TCID50 (median tissue culture infective dose). Mutations in MARV/Ang-GA were identified and compared to sequences of known rodent-adapted MARV/Ang variants, which may benefit future studies characterizing important host adaptation sites in the MARV/Ang viral genome.

Morphological characterization of adult mouse Leydig cells in culture.

The morphology and function of Leydig cells are changed during the development, mature and senility of Leydig cells along the life span of males. This study was to observe the growth morphology of adult mouse Leydig cells in culture, aiming to provide a reference for furthermore understanding of the biological function of Leydig cells by in vitro model. Testes of two-month-old mice were decapsulated and then the Leydig cells were isolated by collagenase digestion and were cultured in DMEM/F12 supplemented with 10% FBS. The Leydig cells were identified by HSD3B staining and RT-PCR. After 48-h Leydig cell culture, both the nucleus and the cytoplasm were very clear under the optical microscope. The nucleus was big and round and the cytoplasm was filled with abundant lipid drops with a strong refractivity. After 5-day culture, Leydig cells were fully elongated in spindle, triangular, polygonal, oval or irregular shapes. Some cells grew in aggregation, and some cells grew independently. Leydig cells in aggregation elongated many cellular tentacles for intercellular connections, which formed an epithelium-like appearance. After HSD3B staining, the individual Leydig cells were stained with different extents, demonstrated that the heterogeneity of HSD3B activity in individual Leydig cells in primary culture. RT-PCR results showed that Leydig cells in culture after 5 days could express Leydig cell-specific transcriptions, HSD3B6, CYP17A1 and StAR. These results showed the morphological characterization of adult mouse Leydig cells in culture, which will lay a foundation to elucidate the relationship between the morphology and function of Leydig cells.

Key Signaling Events for Committing Mouse Pluripotent Stem Cells to the Germline Fate.

The process of germline development carries genetic information and preparatory totipotency across generations. The last decade has witnessed remarkable successes in the generation of germline cells from mouse pluripotent stem cells, especially induced germline cells with the capacity for producing viable offspring, suggesting clinical applications of induced germline cells in humans. However, to date, the culture systems for germline induction with accurate sex-specific meiosis and epigenetic reprogramming have not been well-established. In this study, we primarily focus on the mouse model to discuss key signaling events for germline induction. We review mechanisms of competent regulators on primordial germ cell induction and discuss current achievements and difficulties in inducing sex-specific germline development. Furthermore, we review the developmental identities of mouse embryonic stem cells and epiblast stem cells under certain defined culture conditions as it relates to the differentiation process of becoming germline cells.

Decreased saturated fatty acids, total cholesterol and LDL-C in sdd17 mice.

SDD17, a delta-15 desaturase from the fungus Saprolegnia can convert arachidonic acid to eicosapentanoic acid in yeast, plant embryos, and mammalian cells. Here, we generated transgenic mice that carried two copies of codon-optimized sdd17 cDNA within a non-coding domain of chromosome 6. RT-PCR analysis revealed that the foreign gene was expressed in the transgenic tissues. Gas chromatography showed that the levels of total unsaturated fatty acids in muscle, liver, and spleen tissues were significantly (p<0.05) increased in transgenic mice compared to non-transgenic mice at 3 or 8 weeks of age. In addition, the serum concentrations of total cholesterol and low-density lipoprotein cholesterol in transgenic females, but not in males, were significantly lower than those in sex-matched non-transgenic mice. These results suggest that endogenous sdd17 expression is beneficial for mammalian health and that its effects on fatty acid profiles may differ between sexes.

Mechanism of random integration of foreign DNA in transgenic mice.

Little is known about how foreign DNA is randomly integrated into chromosomes in transgenic animals. In the current study, the insertion sites of 36 transgenic mice were mapped by thermal asymmetric interlaced PCR, and 38 junction sequences were obtained from 30 samples. Analysis of the 38 sequences revealed that 44.7 % of integration events occurred within host gene regions, including 13.2 % (5/38) in exonic regions and 31.6 % (12/38) in intronic regions. The results also revealed that all non-end side integrations of foreign DNA were mediated by short sequence homologies (microhomologies) and that the end side integrations occurred in the presence or absence of microhomologies. In addition, microhomology-mediated mechanisms were also confirmed in four transgenic Arabidopsis thaliana lines. The results indicate that foreign DNA is easily integrated into host gene regions. These results also suggest that the integration of both ends of foreign DNA follows the above-mentioned mechanism in many transgenic/transformed organisms.

[Cell fate switch: lineage reprogramming].

It has been demonstrated that mature cells could switch their fate by the technologies for reprogramming, such as somatic cell nuclear transfer and induced pluripotent stem cells. Recently, this conclusion was further confirmed. It was found that mature differentiated cells could be directly converted into other somatic cells or progenitors with some defined transcription factors. This technology is called lineage reprogramming, which provides an attractive novel alternative to regenerative medicine and animal biotechnology. It is a more convenient and more effective system with less ethical issues. Moreover, lineage reprogramming technology could also facilitate researches on regulation of gene expression. This review highlights the procedures of reprogramming, its characteristics, and significant promise in biomedical applications.

Xuhuai goat H-FABP gene clone, subcellular localization of expression products and the preparation of transgenic mice.

The aim of this study was to clone the heart-type fatty acid binding protein (H-FABP) gene of Xuhuai goat, to explore it bioinformatically, and analyze the subcellular localization using enhanced green fluorescent protein (EGFP). The results showed that the coding sequence (CDS) length of Xuhuai goat H-FABP gene was 402 bp, encoding 133 amino acids (GenBank accession number AY466498.1). The H-FABP cDNA coding sequence was compared with the corresponding region of human, chicken, brown rat, cow, wild boar, donkey, and zebrafish. The similarity were 89%, 76%, 85%, 84%, 93%, 91%, 70%, respectively. For the corresponding amino acid sequences, the similarity were 90%, 79%, 88%, 97%, 95%, 94%, 72%, respectively. This study did not find the signal peptide region in the H-FABP protein; it revealed that H-FABP protein might be a nonsecreted protein. H-FABP expression was detected in vitro by reverse transcription-polymerase chain reaction (RT-PCR), and the EGFP-H-FABP fusion protein was localized to the cytoplasm. The gene could also be transiently and permanently expressed in mice.

[Progress in exploring genes for high fertility in ewes].

Fecundity trait in sheep is regulated by some major genes. Among them, BMPR-IB, BMP-15, and GDF-9 are most distinguishing. The mutant FecB of BMPR-IB has multiplicative effects on ovulation. GDF-9's mutants FecGH, FecI, and BMP-15's mutants FecXI, FecXH, FecXG, FecXB, FecXL, and FecXR increase ovulation rate in the heterozygote but result in sterile phenotypes in the homozygote, while GDF-9's mutant, FecGE, only increases ovulation rate in the homozygote. In addition, Woodlands and Lacaune are known as inheritable major genes. Woodlands gene is an X-linked maternally imprinted gene, and Lacaune is similar to FecB with a multiplicative effect on ovulation rate. The size of the effect of one copy of a mutation on ovulation rate ranges from an extra 0.4 ovulations for the woodlands mutation to an extra 1.5 ovulations for the BMPR-IB and Lacaune mutation. Investigation into these genes will not only help to select breeds with high fertility, but also give a chance to further elucidate the mechanism involved in the phenomenon. This review summaries the source, location, phenotype, and mechanism of the major genes in all breeds of sheep.

[Regulation to curd morphogenesis and genetics of exogenous BoCAL gene in cauliflower].

Cauliflower (Brassica oleracea var. botrytis L.) and cabbage (B. oleracea var capitata L.) are two varieties of species B. oleracea. Cauliflower BobCAL gene losses the original function due to its terminated mutation while cabbage BoCAL gene has the complete function. We obtained cauliflower plants transgenic for BoCAL gene using Arobacterium-mediated transformation. Genetic analysis of T2 generation indicated that all cauliflower plants transgenic for BoCAL failed to form curd and produced the green, loosed inflorescences composed of flower buds instead, demonstrating that cauliflower BobCAL was complemented by cabbage BoCAL, and thus transgenic cauliflower lost the ability to form curd. Selfing of these transgenic plants gave rise to the similar inflorescence phenotype of T3 plants except 15 days delay of inflorescence appearance compared with the plants of T1 generation. When transgenic cauliflower was crossed with wild-type cauliflower, the progenies formed the curd with a few of flower buds, and they delayed inflorescence onset till 135 days after sowing.