Junhua Xiao

The complete mitochondrial genome of Microtus fortis calamorum (Arvicolinae, Rodentia) and its phylogenetic analysis.

Microtus fortis is a special resource of rodent in China. It is a promising experimental animal model for the study on the mechanism of Schistosome japonicum resistance. The first complete mitochondrial genome sequence for Microtus fortis calamorum, a subspecies of M. fortis (Arvicolinae, Rodentia), was reported in this study. The mitochondrial genome sequence of M. f. calamorum (Genbank: JF261175) showed a typical vertebrate pattern with 13 protein coding genes, 2 ribosomal RNAs, 22 transfer RNAs and one major noncoding region (CR region).The extended termination associated sequences (ETAS-1 and ETAS-2) and conserved sequence block 1 (CSB-1) were found in the CR region. The putative origin of replication for the light strand (O(L)) of M. f. calamorum was 35bp long and showed high conservation in stem and adjacent sequences, but the difference existed in the loop region among three species of genus Microtus. In order to investigate the phylogenetic position of M. f. calamorum, the phylogenetic trees (Maximum likelihood and Bayesian methods) were constructed based on 12 protein-coding genes (except for ND6 gene) on H strand from 16 rodent species. M. f. calamorum was classified into genus Microtus, Arvcicolinae for the highly phylogenetic relationship with Microtus kikuchii (Taiwan vole). Further phylogenetic analysis results based on the cytochrome b gene ranged from M. f. calamorum to one of the subspecies of M. fortis, which formed a sister group of Microtus middendorfii in the genus Microtus.

A 1-bp deletion in the γC-crystallin leads to dominant cataracts in mice

To date around 140 genetic alleles have been identified as being responsible for mouse cataract pathology, including Crya, Cryb, Cryg, Maf, Pax6, Pitx3, Sox, Connexins, MIP, and Lim-2. We obtained a dominant cataract mouse model from a spontaneous mutation in the F1 hybrids of outbred strain ICR mice crossed to the inbred strain BALB/cJ mice. Heterozygous and homozygous mutants expressed a nuclear cataract in both eyes. In 8-day-old mice, histological analysis showed that polygon epithelial cells were in the equatorial region and cortex underneath, and vacuole and sponge-like degeneration were in the cortical area underneath the posterior lens capsule. The nucleus of the lens was a deeply stained pink, with the shorter fibers losing their normal arrangement. For the entire eye, there was a blank zone in the equatorial region in 8-day-old mice; however, there was a certain degree of atrophy in cornea tension and retina in the lens in 3-month-old mice. The lens had been serious damaged in the homozygous mutants. For mutation mapping, heterozygous carriers were mated to wild-type C3H/HeJ mice, and offspring (F1 generation) with cataracts were backcrossed to the wild-type C3H/HeJ mice again. N2 mice with cataracts were used for genotyping. Using genome-wide linkage analysis, the mutation was mapped to chromosome 1 and the Cryg gene cluster between two markers was confirmed as the candidate gene. After direct sequencing the cDNA of the Cryg gene cluster, a 1-bp deletion was found in exon 3 of the Crygc gene, leading to a stop codon at the 76th amino acid of exon 3 which results in production of a truncated protein in mutant mice (Leu160Stop). Bioinformatic analysis of the mutant γC-crystallin reveals that the COOH-terminal of the mutant protein deletes a β-sheet, which affects the function of the lens proteins and leads to the development of cataracts.

Effects of Additives on Efficiency and Specificity of Ligase Detection Reaction

Ligase detection reaction (LDR) is adaptable to a wide variety of applications ranging from scientific research to clinical diagnosis, especially in the field of nucleotide polymorphism discrimination and analysis. Efficiency and specificity of LDR are the most two important characteristics that influence its application. To improve the specificity or efficiency of ligase, optimization of the design of LDR probes and the reaction of LDR were investigated previously by most researchers. But the effects of additives on LDR have not been reported. In this study, the effects of additives (DMSO, Tween-20, glycerol, formamide, and PEG-6000) on LDR efficiency and specificity were investigated. The results showed that all of these compounds, except for Tween-20, could improve the specificity of LDR. PEG-6000 was proved to be the best additive among the five tested with an optimal concentration of 5% at which the highest yield was obtained with a relatively improved specificity.

A multiplex sensitive quantification of microRNAs based on competitive PCR

MicroRNAs (miRNAs) are endogenous ~22 nt RNAs that play important regulatory roles in animals and plants by targeting mRNAs for cleavage or gene silencing. Although quantitative real-time PCR (qRT-PCR) had been widely used for miRNAs quantification, a multiplex quantification method is demanding. In this study, we successfully detected 2 miRNAs (miR-505-3p and miR-21a-5p) and an internal control (miR-16-5p) with only one reaction based on competitive PCR (cPCR) with high sensitivity. For each miRNA, two stem-loop reverse transcription (RT) primers were designed to produce two different templates: the competitor cDNA and the target cDNA, which had similar sequences except for 3 nucleotides different in length. RNA from a control sample was reverse transcribed with the competitive RT primers of multiple genes. Samples for test were reverse transcribed with target RT primers to obtain target cDNAs. Target cDNA was mixed with competitor cDNA to be used as the template for a multiplex fluorescent cPCR reaction. The cPCR products were separated on polyacrylamide gel electrophoresis with ABI 377 DNA sequencer and each fluorescent peak was quantified by its intensity. In this method, we compared the expression level of miR-505-3p in two tissues (thalamus and tail) between C57BL/6J and C3H/HeJ mice. The results showed that in the thalamus, which had high abundance of miR-505-3p, both cPCR and SYBR Green based qRT-PCR provided a sensitive quantification outcome. However, in the tail, which had extremely low level of miR-505-3p, it could be steadily detected by cPCR even after 8 times dilution with a relatively high sensitivity, while qRT-PCR can’t detect any product only after 2 times dilution. The variation as low as 12.2% between samples could be clarified by cPCR, which could not be accomplished by qRT-PCR. This method enables multiplex, accurate and sensitive quantification of miRNAs with fewer precious RNA samples than qRT-PCR.

Genome Sequencing of Chromosome 1 Substitution Lines Derived from Chinese Wild Mice Revealed a Unique Resource for Genetic Studies of Complex Traits.

Mouse resources such as Collaborative Cross, outbred stocks, Hybrid Mouse Diversity Panel, and chromosome substitution strains have been instrumental to many progresses in the studies of complex traits genetics. We have established a population of chromosome 1 (Chr 1) substitution lines (C1SLs) in which donor chromosomes were derived from Chinese wild mice. Genome sequencing of 18 lines of this population showed that Chr 1 had been replaced by the donor chromosome. About 4.5 million unique single nucleotide polymorphisms and indels were discovered on Chr 1, of which 1.3 million were novel. Compared with sequenced classical inbred strains, Chr 1 of each C1SL had fivefold more variants, and more loss of function and potentially regulatory variants. Further haplotype analysis showed that the donor chromosome accumulated more historical recombination events, with the largest haplotype block being only 100 kb, and about 57% of the blocks were <1 kb. Subspecies origin analysis showed that these chromosomes had a mosaic genome structure that dominantly originated from Mus musculus musculus and M. m. castaneus subspecies, except for the C57BL/6J-Chr1KM line from M. m. domesticus. In addition, phenotyping four of these lines on blood biochemistry suggested that there were substantial phenotypic variations among our lines, especially line C57BL/6J-Chr1HZ and donor strain C57BL/6J. Further gene ontology enrichment revealed that the differentially expressed genes among liver-expressed genes between C57BL/6J and C57BL/6J-Chr1HZ were enriched in lipid metabolism biological processes. All these characteristics enable C1SLs to be a unique resource for identifying and fine mapping quantitative trait loci on mouse Chr 1, and carrying out systems genetics studies of complex traits.

Comparative quantification of human intestinal bacteria based on cPCR and LDR/LCR.

AIM To establish a multiple detection method based on comparative polymerase chain reaction (cPCR) and ligase detection reaction (LDR)/ligase chain reaction (LCR) to quantify the intestinal bacterial components. METHODS Comparative quantification of 16S rDNAs from different intestinal bacterial components was used to quantify multiple intestinal bacteria. The 16S rDNAs of different bacteria were amplified simultaneously by cPCR. The LDR/LCR was examined to actualize the genotyping and quantification. Two beneficial (Bifidobacterium, Lactobacillus) and three conditionally pathogenic bacteria (Enterococcus, Enterobacterium and Eubacterium) were used in this detection. With cloned standard bacterial 16S rDNAs, standard curves were prepared to validate the quantitative relations between the ratio of original concentrations of two templates and the ratio of the fluorescence signals of their final ligation products. The internal controls were added to monitor the whole detection flow. The quantity ratio between two bacteria was tested. RESULTS cPCR and LDR revealed obvious linear correlations with standard DNAs, but cPCR and LCR did not. In the sample test, the distributions of the quantity ratio between each two bacterial species were obtained. There were significant differences among these distributions in the total samples. But these distributions of quantity ratio of each two bacteria remained stable among groups divided by age or sex. CONCLUSION The detection method in this study can be used to conduct multiple intestinal bacteria genotyping and quantification, and to monitor the human intestinal health status as well.

Improved PCR-BSP Assay for Multiplex Methylation Pattern Analysis in Minimal Amount of DNA

Cell-specific DNA methylation pattern detection is of great importance for the tumorigenesis and differentiation studies. Comparatively, large amounts of DNA were needed for traditional methods of DNA methylation pattern detection, and therefore, more sensitive method for high throughput analysis with a limited amount of DNA is needed. With Mouse 3T3 cells, we developed new multiplex-nested PCR technologies for bisulfite-assisted genomic sequencing PCR (BSP) methylation pattern detection method. Primers step add-in method and templates precipitation methods efficiently increase the throughput of the assay, and the nested PCR method also increased the sensitivity. The optimized assay could successfully detect 15 sequences of methylation pattern with a minimal amount of DNA (500–1,000 cells of genome DNA).

Dissection of the maternal effects on puberty onset by embryo transplantation in mouse

Puberty onset in mammals is affected by multiple genetic and environmental factors. Among which, the maternal effect could have played a considerable role. In our previous study, we found that the F1 offspring from reciprocal crosses between C3H/HeJ (C3H) and C57BL/6J (B6) mice differed significantly in the timing of puberty in both sexes, though they had identical genomic background. In order to dissect the causative factors to such phenomenon of maternal effect, embryos from reciprocal crosses of C3H/HeJ and C57BL/6J mice were collected and transplanted to the uterus of either strain of mothers, and the puberty onset of pups were compared between different recipient mothers and egg origins. The results showed that the male pups from C3H recipient mothers attained puberty onset earlier than those from B6 recipients significantly. while the female pups did not show such difference. On the other hand, the egg origin made no difference in the puberty onset of either sex, yet it influenced the birth weight of female pups significantly (p<0.05). The manipulation of embryo transplantation delayed the puberty onset of pups dramatically. A mitochondria substitution strain between B6 and C3H (BmC), which had the genome background of B6 and a mitochondrial hyplotype of C3H, had the same phenotype of puberty onset as B6. The integrated results indicated that the uterine environment was the major causative factors to the maternal effect on the differential puberty onset in reciprocal crosses of F1 hybrids between B6 and C3H mice.

MiR-505-3p, a New Repressor of Puberty Onset in Female Mice

Puberty onset is a complex trait regulated by multiple genetic and environmental factors. In this study, we narrowed a puberty-related QTL on chromosome X in female mice to a 1.7 Mb region and deduced that miR-505-3p was the functional gene. In a GT1-7 cell line with stable overexpression of miR-505-3p (pGT1-7), both ribosomes and ribosome biogenesis pathways were affected, and the expression of some puberty-related genes was down-regulated. The amount of mRNA and protein products of the Srsf1 gene decreased by 50 percent, and the expression of puberty-related genes was rescued by the overexpression of Srsf1. With the down regulation of Srsf1 expression through shRNA, the mRNA accumulation of puberty-related genes decreased simultaneously in the GT1-7 cell line. The results of RIP-seq showed that SF2, the protein of the Srsf1 gene, primarily bound ribosome protein (RP) mRNAs in GT1-7 cells. miR-505-3p knockout female mice showed earlier vaginal opening, higher serum gonadotrophin levels and higher expression of puberty-related and Srsf1 genes in the hypothalamus than their wild-type littermates. B6 female mice with ectopic expression of miR-505-3p in the hypothalamus showed significant growth retardance and later VO than wild types. These results suggest that miR-505-3p may regulate puberty onset via the Srsf1 gene and RP expression, which reveals a new regulatory pathway in mammalian puberty onset involving microRNA, SF2 and ribosome proteins. Author summary In this study, we identified miR-505-3p in a puberty-related QTL on the X chromosome as a female puberty onset repressor using a positional cloning strategy. GT1-7 cell lines stably overexpressing miR-505-3p (pGT1-7) showed Kiss1 and GnRH down-regulation. We also identified Srsf1 as the functional target gene of miR-505-3p in GT1-7 cells. Compared to wild-type mice, miR-505-3p knockout female mice showed puberty onset four days sooner, along with the overexpression of miR-505-3p in the hypothalamus 2 days later. Thus, miR-505-3p is a new repressor of puberty onset in female mice.

MiR-505-3p is a Repressor of the Puberty Onset in Female Mice

Puberty onset is a complex trait regulated by multiple genetic and environmental factors. In this study, we narrowed a puberty related QTL down to a 1.7 Mb region on chromosome X in female mice and inferred miR-505-3p as the functional gene. We conducted ectopic expression of miR-505-3p in the hypothalamus of prepubertal female mice through lentivirus-mediated orthotopic injection. The impact of miR-505-3p on female puberty was evaluated by the measurement of pubertal events and histological analysis. The results showed that female mice with overexpression of miR-505-3p in the hypothalamus manifested later puberty onset timing both in vaginal opening and ovary maturation, followed by weaker fertility lying in the longer interval time between mating and delivery, higher abortion rate and smaller litter size. We also constructed miR-505-3p knockout mice by CRISPR/Cas9 technology. MiR-505-3p knockout female mice showed earlier vaginal opening timing, higher serum gonadotrophin and higher expression of puberty-related gene, as well as its target gene Srsf1 in the hypothalamus than their wild type littermates. Srsf1 was proved to be the target gene of miR-505-3p that played the major role in this process. The results of RIP-seq (RNA Immunoprecipitation-sequencing) showed that SF2, the protein product of Srsf1 gene, mainly bound to ribosome protein (RP) mRNAs in GT1-7 cells. The collective evidence implied that miR-505-3p/SRSF1/RP could play a role in the sexual maturation regulation of mammals. Author summary The puberty onset in mammals is a vital biological process that signals the acquisition of reproductive capacity. The initiation of puberty is triggered by the activation of hypothalamic pulsatile GnRH surge. The dysregulation of pubertal development shows relevance to later health risks of type 2 diabetes, cardiovascular disease, breast cancer and other health disorders. Recent progress indicates that a lot of genes play a role in the excitatory or inhibitory regulation of GnRH release. However, the detailed pathway of pubertal timing remains unclear. Our previous studies isolated an X-linked QTL that was associated with the timing of puberty in mice. In this study, we proved that miR-505-3p was a female puberty onset regulator based on data from positional cloning, ectopic expression and knockout mouse models. We also assigned Srsf1 as the functional target gene of miR-505-3p underlying this process. The results of RIP-seq showed that SF2, the protein of Srsf1 gene, preferential bound to ribosome protein (RP) mRNAs in GT1-7 cells. We propose that miR-505-3p/SF2/RP could play a role in the sexual maturation regulation of mammals.