[Studies on the transcriptomes of non-model organisms].

The transcriptome represents the whole complement of RNA transcripts in cells or tissues and reflects the expressed genes at various life stages, tissue types, physiological states, and environmental conditions. Transcriptome analysis provides a comprehensive understanding of gene expression and its regulation. Non-model organism has many interesting traits of which model organisms lack, and the study of its transcriptome has great significance in solving the questions of genetic evolution, genetic breeding, ecology and so on. Because of absence of reference genome information, and traditional transcriptome research methods which are complicated to operate, long experimental period and costly, slow progress has been made in the research of non-model organism transcriptome. Fortunately, RNA sequencing (RNA-seq), the next-generation sequencing technology, has completely changed the way of transcriptome study, becoming an advanced technology on the investigation of non-model organism transcriptome. In this paper, we give a summary of the non-model organism transcriptome research using RNA-seq in recent years and briefly describe its general flow and principles from aspects of sample preparation, high throughput DNA sequencing, and bioinformatics analysis. Finally, questions that are still open and awaiting further research are also discussed.

Development of cloned embryos from porcine neural stem cells and amniotic fluid-derived stem cells transfected with enhanced green fluorescence protein gene.

We assessed the developmental ability of embryos cloned from porcine neural stem (NS) cells, amniotic fluid-derived stem (AFS) cells, fetal fibroblast cells, adult fibroblast, and mammary gland epithelial cells. The five cell lines were transfected with enhanced green fluorescence protein gene respectively using lipofection. NS and AFS cells were induced to differentiate in vitro. Stem cells and their differentiated cells were harvested for analysis of the markers using RT-PCR. The five cell lines were used for nuclear transfer. The two-cell stage-cloned embryos derived from each cell line were transferred into the oviducts of surrogate mothers. The results showed that both NS and AFS cells expressed POU5F1, THY1 and SOX2, and they were both induced to differentiate into astrocyte (GFAP+), oligodendrocyte (GalC+), neuron (NF+, ENO2+, and MAP2+), adipocyte (LPL+ and PPARG-D+), osteoblast (osteonectin+ and osteocalcin+), myocyte (MYF6+ and MYOD+), and endothelium (PECAM1+, CD34+, CDH5+, and NOS3+) respectively. Seven cloned fetuses (28 days and 32 days) derived from stem cells were obtained. The in vitro developmental ability (morula-blastocyst rate was 28.26-30.07%) and in vivo developmental ability (pregnancy rate were 1.67-2.17%) of the embryos cloned from stem cells were higher (P<0.05) than that of the embryos cloned from somatic cells (morula-blastocyst rate was 16.27-19.28% and pregnancy rate was 0.00%), which suggests that the undifferentiated state of the donor cells increases cloning efficiency.

Developmental expression and possible functional roles of mouse Nlrp4e in preimplantation embryos.

Increasing evidence suggests that some Nlrp genes are crucial for oogenesis, folliculogenesis, and early embryonic development. Nlrp4e is one of seven copies of Nlrp4, which plays a putative role in the reproduction system in mice. Gene duplication is regarded as an important driving force behind the evolution of novel genes with new or altered functions. We investigated the role of Nlrp4e in oocyte and preimplantation embryos by determining its expression profile using quantitative real-time polymerase chain reaction. Nlrp4e mRNA accumulated during oogenesis. Moreover, Nlrp4e transcripts were upregulated during the two-cell stage and then declined sharply and became almost undetectable, which represents a crucial time for major embryonic genome activation in the mouse. Knockdown of Nlrp4e in fertilized eggs using RNA interference resulted in arrested development between the two- and eight-cell stages in a dose-dependent manner. However, targeted inhibition of Nlrp4e in germinal-vesicle-stage oocytes had no phenotypic effects on oocyte maturation. The above experiments were also carried out in parthenogenetic embryos to determine the effects of Nlrp4e in embryos without a paternal genome. The results of this study indicate that Nlrp4e, a maternal-zygotic-effect gene, may not be involved in oocyte maturation but may play a critical role in early embryogenesis.

Effects of the removal of cytoplasm on the development of early cloned bovine embryos.

Oocyte cytoplasm plays a prominent role in cloned embryonic development. To investigate the influence of oocyte cytoplasmic amount on cloned embryo development, we generated bovine somatic cell nuclear transfer (SCNT) embryos containing high (30-40% of the cytoplasm was removed), medium (15-25% of the cytoplasm was removed) and low (<10% of the cytoplasm was removed) nucleocytoplasmic volume ratios (N/C) using enucleated metaphase II oocyte as recipient, and fibroblast as donor nucleus, and analyzed the expression levels of ND1, Cytb and ATPase6, as well as the embryonic quality. The results indicated: (1) the process of embryonic development was not influenced by <40% of cytoplasm removal; (2) the rate of blastocyst formation, the total number of blastomere and the ratio of ICM to TE were inversely proportional to the N/C; (3) SCNT embryos with reduced volume equal to 75-85% or >90% of an intact oocyte volume showed similar karyotype structure of the donor cells; (4) the number of mtDNA copy was larger in low N/C embryos than that in medium or high N/C embryos, and the expression levels of each gene hardly varied from the 2-cell to 8-cell stage, while the expression levels increased dramatically at the blastocyst stage; (5) from 16-cell to the blastocyst stage, the change of the expression level of each gene was not significant between low N/C embryos and IVF embryos, but it was more significant than those of high or medium N/C embryos. The results suggest that the decrease of mtDNA copy number and mitochondrial gene expression may be related to the impairment in early embryonic development, and removal of <10% adjacent cytoplasm volume may be optimal for bovine SCNT embryo development.

Comparation of enhanced green fluorescent protein gene transfected and wild-type porcine neural stem cells.

The aim of this study was to transfect and express the enhanced green fluorescence protein (EGFP) gene into porcine neural stem cells (NSCs) to determine whether EGFP can be used as a marker to monitor NSCs. NSCs were isolated from embryonic day 30 fetal pig brain and transfected with EGFP gene using lipofection. Transfected and wild-type NSCs were induced to differentiate into cells of neuronal and myogenic lineages. Markers of passage three NSCs and their differentiated cells were tested by reverse transcription polymerase chain reaction. The results showed that EGFP could be expressed in NSCs and the differentiated cells. NSCs expressed Nestin, NogoA, DCX, Hes1, Oct4, CD-90 and Sox2. NSCs could differentiated into astrocyte (GFAP(+)), oligodendrocyte (GalC(+)), neuron (NF(+), NSE(+) and MAP2(+)) and myocyte (myf-6(+) and myoD(+)). We concluded that EGFP can be used as a marker in monitoring NSCs.

[Optimization of culture measure for bovine-bovine and goat-bovine cloned embryos in vitro].

UNLABELLED: This study is conducted to explore an effective culture method for supporting the embryo development. The cattle fetal ear fibroblasts and the goat fetal ear fibroblasts are transplanted into the enucleated cattle oocytes separately by oocyte intraplasmic nuclear injection method to construct bovine cloned embryos and goat-bovine cloned embryos. The embryos are first cultivated in modified charles rosenkrans 2 amino acid medium (mCR2aa) and modified synthetic oviduct fluid medium (mSOF) separately. Then BSA (8 mg/mL) or FBS (10%) can be added to mSOF according to the different culture period. The supplements and orders, added during the first three days and after three days are as follow: BSA and BSA, BSA and FBS, FBS and BSA, FBS and FBS. On the basis of the cleavage rate, 8/16-cell rate, blastocysts rate and total cell number of blastocysts, the best culture way can be screened out. RESULT: First, cleavage rate, 8/16-cell rate, blastocysts rate and total cell number of blastocysts, cultivated in mSOF solution are all higher than those cultivated in mCR2aa( P < 0.05). Second, the cleavage rate and 8/16-cell rate, adding BSA and FBS into mSOF, are in turn 79.8% +/- 7.1%, 49.7% +/- 3.5%, 21.5% +/- 1.8%, and 115.2 +/- 4.3 in bovine cloned embryo, and 40.1% +/- 6.3%, 29.2% +/- 2.0%, 13.4% +/- 2.1% and 100.1 +/- 3.0 in goat-bovine cloned embryo, which are significant higher than other culture groups (P < 0.05). CONCLUSION: The goat-bovine cloned embryo can be cultivated by the optimized culture measure of bovine cloned embryo. The best culture ways of bovine cloned embryo and goat-bovine cloned embryo are all to use mSOF supplemented BSA in the first three days and then use mSOF supplemented FBS in the next five days.