Selection of Reliable Reference Genes for Gene Expression Studies on Rhododendron molle G. Don.

The quantitative real-time polymerase chain reaction (qRT-PCR) approach has become a widely used method to analyze expression patterns of target genes. The selection of an optimal reference gene is a prerequisite for the accurate normalization of gene expression in qRT-PCR. The present study constitutes the first systematic evaluation of potential reference genes in Rhododendron molle G. Don. Eleven candidate reference genes in different tissues and flowers at different developmental stages of R. molle were assessed using the following three software packages: GeNorm, NormFinder, and BestKeeper. The results showed that EF1-α (elongation factor 1-alpha), 18S (18s ribosomal RNA), and RPL3 (ribosomal protein L3) were the most stable reference genes in developing rhododendron flowers and, thus, in all of the tested samples, while tublin (TUB) was the least stable. ACT5 (actin), RPL3, 18S, and EF1-α were found to be the top four choices for different tissues, whereas TUB was not found to favor qRT-PCR normalization in these tissues. Three stable reference genes are recommended for the normalization of qRT-PCR data in R. molle. Furthermore, the expression profiles of RmPSY (phytoene synthase) and RmPDS (phytoene dehydrogenase) were assessed using EF1-α, 18S, ACT5, RPL3, and their combination as internals. Similar trends were found, but these trends varied when the least stable reference gene TUB was used. The results further prove that it is necessary to validate the stability of reference genes prior to their use for normalization under different experimental conditions. This study provides useful information for reliable qRT-PCR data normalization in gene studies of R. molle.

Association of vitamin D deficiency and frailty: A systematic review and meta-analysis.

There is a biologically plausible association between low vitamin D, specifically serum 25-hydroxyvitamin D [25(OH)D] level, and frailty. We conducted a systematic review and meta-analysis to describe the association between low 25(OH)D level and frailty. We searched literature in OVID (Medline), EMBASE, Web of Knowledge and Cochrane CENTRAL Library Issue in May 2016, for cohort studies evaluating association of low 25(OH)D level with the risk of frailty. Studies were reviewed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses(PRISMA) guidelines. A total of seven studies(17,815 participants)were eligible in our study. The prevalence of frailty ranged from 3.9% to 31.9%. The pooled OR of frailty for the lowest versus the highest level of vitamin D was 1.27 (95% CI=1.17-1.38, I2=59%), suggesting that low level of vitamin D was significantly associated with the risk of frailty. In addition, results of subgroups analysis indicated that low level of vitamin D was significantly associated with the risk of frailty in female (pooled OR=1.27, 95% CI=1.15-1.40). Similar result was also found when frailty was defined by the Fried criteria or the modified Fried criteria (pooled OR=1.25, 95% CI=1.14-1.37), and FRAIL scale (pooled OR=1.55, 95% CI=1.07-2.25). Compared to the highest level of 25(OH)D, the association between frailty and the lowest level of 25(OH)D was significant in our study.

Critical developmental stages for the efficiency of somatic cell nuclear transfer in zebrafish.

Somatic cell nuclear transfer (SCNT) has been performed extensively in fish since the 1960s with a generally low efficiency of approximately 1%. Little is known about somatic nuclear reprogramming in fish. Here, we utilized the zebrafish as a model to study reprogramming events of nuclei from tail, liver and kidney cells by SCNT. We produced a total of 4,796 reconstituted embryos and obtained a high survival rate of 58.9-67.4% initially at the 8-cell stage. The survival rate exhibited two steps of dramatic decrease, leading to 8.7-13.9% at the dome stage and to 1.5-2.96% by the shield stage. Concurrently, we observed that SCNT embryos displayed apparently delayed development also at the two stages, namely the dome stage (1:30 ± 0:40) and the shield stage (2:50 ± 0:50), indicating that the dome and shield stage are critical for the SCNT efficiency. Interestingly, we also revealed that an apparent alteration in klf4 and mycb expression occurred at the dome stage in SCNT embryos from all the three donor cell sources. Taken together, these results suggest that the dome stage is critical for the SCNT efficiency, and that alternated gene expression appears to be common to SCNT embryos independently of the donor cell types, suggesting that balanced mycb and klf4 expression at this stage is important for proper reprogramming of somatic nuclei in zebrafish SCNT embryos. Although the significant alteration in klf4 and mycb expression was not identified at the shield stage between ZD and SCNT embryos, the importance of reprogramming processes at the shield stage should not be underestimated in zebrafish SCNT embryos.

Identification of differentially expressed genes between cloned and zygote-developing zebrafish (Danio rerio) embryos at the dome stage using suppression subtractive hybridization.

Comparative analyses of differentially expressed genes between somatic cell nuclear transfer (SCNT) embryos and zygote-developing (ZD) embryos are important for understanding the molecular mechanism underlying the reprogramming processes. Herein, we used the suppression subtractive hybridization approach and from more than 2900 clones identified 96 differentially expressed genes between the SCNT and ZD embryos at the dome stage in zebrafish. We report the first database of differentially expressed genes in zebrafish SCNT embryos. Collectively, our findings demonstrate that zebrafish SCNT embryos undergo significant reprogramming processes during the dome stage. However, most differentially expressed genes are down-regulated in SCNT embryos, indicating failure of reprogramming. Based on Ensembl description and Gene Ontology Consortium annotation, the problems of reprogramming at the dome stage may occur during nuclear remodeling, translation initiation, and regulation of the cell cycle. The importance of regulation from recipient oocytes in cloning should not be underestimated in zebrafish.

Identification and characterization of a novel gene differentially expressed in zebrafish cross-subfamily cloned embryos.

BACKGROUND: Cross-species nuclear transfer has been shown to be a potent approach to retain the genetic viability of a certain species near extinction. However, most embryos produced by cross-species nuclear transfer were compromised because that they were unable to develop to later stages. Gene expression analysis of cross-species cloned embryos will yield new insights into the regulatory mechanisms involved in cross-species nuclear transfer and embryonic development. RESULTS: A novel gene, K31, was identified as an up-regulated gene in fish cross-subfamily cloned embryos using SSH approach and RACE method. K31 complete cDNA sequence is 1106 base pairs (bp) in length, with a 342 bp open reading frame (ORF) encoding a putative protein of 113 amino acids (aa). Comparative analysis revealed no homologous known gene in zebrafish and other species database. K31 protein contains a putative transmembrane helix and five putative phosphorylation sites but without a signal peptide. Expression pattern analysis by real time RT-PCR and whole-mount in situ hybridization (WISH) shows that it has the characteristics of constitutively expressed gene. Sub-cellular localization assay shows that K31 protein can not penetrate the nuclei. Interestingly, over-expression of K31 gene can cause lethality in the epithelioma papulosum cyprinid (EPC) cells in cell culture, which gave hint to the inefficient reprogramming events occurred in cloned embryos. CONCLUSION: Taken together, our findings indicated that K31 gene is a novel gene differentially expressed in fish cross-subfamily cloned embryos and over-expression of K31 gene can cause lethality of cultured fish cells. To our knowledge, this is the first report on the determination of novel genes involved in nucleo-cytoplasmic interaction of fish cross-subfamily cloned embryos.

Cloning and characterization of cytochrome c oxidase subunit I (COXI) in Gobiocypris rarus.

In study of gene expression profile in cloned embryos which derived from D. rerio embryonic nuclei and G. rarus enucleated eggs, cytochrome c oxidase subunit I (COXI) of G. rarus, exhibiting difference at expression level between cloned embryos and zebrafish embryo, was cloned. Its full cDNA length is 1654 bp and contains a 1551 bp open reading frame, encoding a 5.64 kDa protein of 516 amino acids. The alignment result shows that mitochondrion tRNAser is co-transcripted with COXI, which just was the 3'-UTR of COXI. Molecular phylogenic analysis based on COXI indicates G. rarus should belong to Gobioninae, which was not in agreement with previous study according to morphological taxonomy. Comparison of DNA with cDNA shows that RNA editing phenomenon does not occur in the COXI of G. rarus.

Efficient RNA interference in zebrafish embryos using siRNA synthesized with SP6 RNA polymerase.

Double-stranded RNA (dsRNA) has been shown to be a useful tool for silencing genes in zebrafish (Danio rerio), while the blocking specificity of dsRNA is still of major concern for application. It was reported that siRNA (small interfering RNA) prepared by endoribonuclease digestion (esiRNA) could efficiently silence endogenous gene expression in mammalian embryos. To test whether esiRNA could work in zebrafish, we utilized Escherichia coli RNaseIII to digest dsRNA of zebrafish no tail (ntl), a mesoderm determinant in zebrafish and found that esi-ntl could lead to developmental defects, however, the effective dose was so close to the toxic dose that esi-ntl often led to non-specific developmental defects. Consequently, we utilized SP6 RNA polymerase to produce si-ntl, siRNA designed against ntl, by in vitro transcription. By injecting in vitro synthesized si-ntl into zebrafish zygotes, we obtained specific phenocopies of reported mutants of ntl. We achieved up to a 59%no tail phenotype when the injection concentration was as high as 4 microg/microL. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization analysis showed that si-ntl could largely and specifically reduce mRNA levels of the ntl gene. As a result, our data indicate that esiRNA is unable to cause specific developmental defects in zebrafish, while siRNA should be an alternative for downregulation of specific gene expression in zebrafish in cases where RNAi techniques are applied to zebrafish reverse genetics.

Cytoplasmic impact on cross-genus cloned fish derived from transgenic common carp (Cyprinus carpio) nuclei and goldfish (Carassius auratus) enucleated eggs.

In previous studies of nuclear transplantation, most cloned animals were obtained by intraspecies nuclear transfer and are phenotypically identical to their nuclear donors; furthermore, there was no further report on successful fish cloning since the report of cloned zebrafish. Here we report the production of seven cross-genus cloned fish by transferring nuclei from transgenic common carp into enucleated eggs of goldfish. Nuclear genomes of the cloned fish were exclusively derived from the nuclear donor species, common carp, whereas the mitochondrial DNA from the donor carp gradually disappeared during the development of nuclear transfer (NT) embryos. The somite development process and somite number of nuclear transplants were consistent with the recipient species, goldfish, rather than the nuclear donor species, common carp. This resulted in a long-lasting effect on the vertebral numbers of the cloned fish, which belonged to the range of goldfish. These demonstrate that fish egg cytoplasm not only can support the development driven by transplanted nuclei from a distantly related species at the genus scale but also can modulate development of the nuclear transplants.

[Cloning and expression analysis in mature individuals of salmon gonadotropin-releasing hormone (sGnRH) gene in common carp].

Two types of complementary DNAs (cDNA) encoding the precursor of salmon gonadotropin-releasing hormone (sGnRH, [Trp7, Leu8] GnRH) are cloned and sequenced from common carp brain using rapid amplification of cDNA ends (RACE). The two cDNAs are referred to sGnRH cDNA1 and cDNA2, and the full-length fragment of cDNA1 and cDNA2 were 393 and 478 bp, respectively. Two sGnRH cDNAs contain an open reading frame of 285 bp, which encodes the sGnRH precursor including 94 amino acid residues. The sGnRH precursors consist of a signal peptide, sGnRH decapeptide and a GnRH-associated peptide (GAP) which is linked by the processing site (Gly-Lys-Arg). Two different sGnRH genes are characterized by intron trapping, and they share a similar structure composed of four exons and three introns. The nucleotide sequences identity of intron 1, intron 2 and intron 3 in sGnRH gene1 and gene2 are 71.1, 76. 1 and 88.0%, respectively. The basic structure and encoding architecture of sGnRH cDNAs and genes are similar with the reported GnRH. It is presumed that all the GnRH variants may evolve from a common ancestral molecular. Southern blot results confirm further the conclusion that there are two differential sGnRH genes in common carp genome. The results of reverse transcription-polymerase chain reaction (RT-PCR) assaying show that the two sGnRH genes co-express in the dissected brain regions, pituitary and ovary with the exception of expression of sGnRH genes in testis. According to the co-existence of two sGnRH genes in distinct brain regions,pituitary and ovary, it is presumed that sGnRH plays an important role in the regulation of hypothalamic-pituitary-gonadal (HPG) axis,and that sGnRH also operates as the neuromodulator, autocrine and/or paracrine regulator.

[Studies of F1 of transgenic allotetraploid hybrids of Carassius auratus red var. (Female) x Cyprinus carpio (Male)].

The tetraploid fish has been developed by assortative breeding the hybrids of Carassius auratus red var. (Female) x cyprinus carpio (Male), which has the stable genetic characters and can reproduce themselves. An "all-fish" recombinant DNA construct (pCAgcGHc) containing common carp beta-actin gene promoter and cDNA for grass carp (Ctenopharyngodon idella) growth hormone gene was introduced into fertilized eggs of the allotetroploid fish through microinjection as soon as artificial insemination was done. Artificial insemination was carried out between the female and the male transgenic allotetraploid fish which contain the "all-fish" recombinant DNA construct (pCAgcGHc) and are the biggest in the size. Fifty F1 samples of transgenic allotetraploid fish of 150 days and 50 allotetraploid fish (regarded as the control) were chosen, and the weight and the body length of each were measured, the results showed that F1 of transgenic allotetraploid fish of 150 days had obvious growth dominance compared with the control. Genomic DNA of tail fin was extracted from 20 F1 of transgenic allotetraploid fish of 150 days and the control. Proper primers were introduced to check whether the sample had the transgene. Pa, the upstream primer, is located in beta-actin promoter, and Pg, the downstream primer, is located in growth hormone cDNA for grass carp (gcGHc). The transgene was detected in 90% F1 of transgenic allotetraploid fish in tail fin DNA by polymerase chain reaction (PCR) amplification. Sperm could be squeezed out from a few F1 of transgenic allotetraploid fish of 150 days, however, this phenomenon did not exist in the controls. The importance of forming the pure line of transgenic allotetraploid was elucidate in the paper.