The emu is a novel poultry species in Japan. However, Japanese farmed emu populations have reduced genetic diversity owing to inbreeding. We have previously suggested that there are genetic resources ...in the Tohoku Safari Park (TSP) and Fuji/Kakegawa Kachoen Garden Park (FGP/KGP) to extend the genetic diversity of commercial emu farms based on microsatellite (SSR) and mitochondrial DNA. However, those markers provide relatively poor information. Thus, we investigated the genetic structure of farmed Japanese populations based on a large-scale genotyping system using RAD-seq and verified the usefulness of TSP and FGP/KGP as genetic resources for expanding genetic diversity. Admixture, phylogenetic, and principal component analyses based on 28,676 SNPs showed that TSP individuals were ancestors in the Okhotsk Emu Farm (OEF). FGP/KGP individuals showed a unique genetic component that differed from that of the others. We have previously reported that the mitochondrial haplotypes of FGP/KGP were shared with an isolated wild population in eastern Australia. These results suggest that FGP/KGP individuals originated from an eastern Australia isolated population different from other populations including ancestral of OEF/TSP. Our results would provide information for the development of Japanese emu farms and industry and for the conservation of genetic resources in the Australian wild emu.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
We present a robust method called improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) that delivers CRISPR ribonucleoproteins to E0.7 embryos via in situ electroporation. The ...method generates mouse models containing single-base changes, kilobase-sized deletions, and knock-ins. The efficiency of i-GONAD is comparable to that of traditional microinjection methods, which rely on ex vivo handling of zygotes and require recipient animals for embryo transfer. In contrast, i-GONAD avoids these technically difficult steps, and it can be performed at any laboratory with simple equipment and technical expertise. Further, i-GONAD-treated females retain reproductive function, suggesting future use of the method for germline gene therapy.
Microinjection is considered the gold standard technique for delivery of nucleic acids (NAs; transgenes or genome editing tools such as CRISPR/Cas9 systems) into embryos, for creating genetically ...modified organisms. It requires sophisticated equipment as well as well-trained and highly skilled personnel to perform the micro-injection technique. Here, we describe a novel and simple microinjection-independent technique, called Genome-editing via Oviductal Nucleic Acids Delivery (GONAD). Using GONAD, we show that NAs (e.g., eGFP mRNA or Cas9 mRNA/sgRNAs) can be effectively delivered to pre-implantation embryos within the intact mouse oviduct by a simple electroporation method, and result in the desired genetic modification in the embryos. Thus GONAD can bypass many complex steps in transgenic technology such as isolation of zygotes, microinjection of NAs into them, and their subsequent transfer to pseudo-pregnant animals. Furthermore, this method can potentially be used for genome editing in species other than mice.
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Fibroblast growth factor 5 (FGF5) is an important molecule required for the transition from anagen to catagen phase of the mammalian hair cycle. We previously reported that Syrian hamsters harboring ...a 1-bp deletion in the Fgf5 gene exhibit excessive hair growth in males. Herein, we generated Fgf5 mutant mice using genome editing via oviductal nucleic acid delivery (GONAD)/improved GONAD (i-GONAD), an in vivo genome editing system used to target early embryos present in the oviductal lumen, to study gender differences in hair length in mutant mice. The two lines (Fgf5go-malc), one with a 2-bp deletion (c.552_553del) and the other with a 1-bp insertion (c.552_553insA) in exon 3 of Fgf5, were successfully established. Each mutation was predicted to disrupt a part of the FGF domain through frameshift mutation (p.Glu184ValfsX128 or p.Glu184ArgfsX128). Fgf5go-malc1 mice had heterogeneously distributed longer hairs than wild-type mice (C57BL/6J). Notably, this change was more evident in males than in females (p < 0.0001). Immunohistochemical analysis revealed the presence of FGF5 protein in the dermal papilla and outer root sheath of the hair follicles from C57BL/6J and Fgf5go-malc1 mice. Histological analysis revealed that the prolonged anagen phase might be the cause of accelerated hair growth in Fgf5go-malc1 mice.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Characterization of carcass traits and fat quality is important to effectively produce and genetically improve emus. We investigated carcass traits in 309 emus. The meat production of female emus ...showed a significantly higher value than that of males (P < 0.01). The fat weight of male (9.232 ± 3.156 kg) was larger than that of the female (7.772 ± 2.697 kg). The fat yield (fat weight per kg of body weight) was strongly correlated to body weight (r = 0.79 and r = 0.75 in male and female, respectively). The fat melting points of females and males were 19.19 ± 3.39°C and 19.39 ± 3.39°C, respectively, without significant difference. Since the fat melting point did not correlate to body and fat weights, we predicted that it was an independent trait from body growth and was highly influenced by genetic elements. Percentages of palmitic, stearic, oleic, linoleic, and α-linolenic acids were 22.27 ± 3.50%, 9.37 ± 1.90%, 54.11 ± 5.17%, 13.54 ± 7.80% and 0.71 ± 0.59%, respectively. Among them, linoleic acid contents showed a wide individual difference (range 0.3–19.9%). The oleic/stearic acid ratio showed a negative correlation to the fat melting point. These results suggest that the fat melting point is a good indicator of C18:1/C18:0 ratio in emu fat.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Visual impairment leads to a decrease in quality of life. Cataract is the most commonly observed ocular disease in humans that causes vision disorders. The risk factors associated with cataract ...development include aging, infections, eye injuries, environmental causes, such as radiation and exposure to ultraviolet rays in sunlight, and genetic mutations. Additionally, several cataract patients display phenotypic heterogeneity, suggesting the role of genetic modifiers in the modulation of severity and onset time of cataractogenesis. However, the genetic modifiers associated with cataract have not been identified in humans yet. In contrast, the identification and mapping of genetic modifiers have been successfully carried out in mice and rats. In this review, we focus on the genetic modifiers of cataract in the rodent models.
While the study of phenotypic variation is a central theme in evolutionary biology, the genetic approaches available to understanding this variation are usually limited because of a lack of genomic ...information in non-model organisms. This study explored the utility of next-generation sequencing (NGS) technologies for studying phenotypic variations between 2 populations of a non-model species, the Hokkai shrimp (Pandalus latirostris; Decapoda, Pandalidae). Before we performed transcriptome analyses using NGS, we examined the genetic and phenotypic differentiation between the populations. Analyses using microsatellite DNA markers suggested that these populations genetically differed from one another and that gene flow is restricted between them. Moreover, the results of our 4-year field observations indicated that the egg traits varied genetically between the populations. Using mRNA extracted from the ovaries of 5 females in each population of Hokkai shrimp, we then performed a transcriptome analysis of the 2 populations. A total of 13.66 gigabases (Gb) of 75-bp reads was obtained. Further, 58,804 and 33,548 contigs for the first and second population, respectively, and 47,467 contigs for both populations were produced by de novo assembly. We detected 552 sequences with the former approach and 702 sequences with the later one; both sets of sequences showed greater than twofold differences in the expression levels between the 2 populations. Twenty-nine sequences were found in both approaches and were considered to be differentially expressed genes. Among them, 9 sequences showed significant similarity to functional genes. The present study showed a de novo assembly approach for the transcriptome of a non-model species using only short-read sequence data, and provides a strategy for identifying sequences showing significantly different expression levels between populations.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In breast cancer cells, the metastatic cell state is strongly correlated to epithelial-to-mesenchymal transition (EMT) and the CD44+/CD24- stem cell phenotype. However, the MCF-7 cell line, which has ...a luminal epithelial-like phenotype and lacks a CD44+/CD24- subpopulation, has rare cell populations with higher Matrigel invasive ability. Thus, what are the potentially important differences between invasive and non-invasive breast cancer cells, and are the differences related to EMT or CD44/CD24 expression?
Throughout the sequential selection process using Matrigel, we obtained MCF-7-14 cells of opposite migratory and invasive capabilities from MCF-7 cells. Comparative analysis of epithelial and mesenchymal marker expression was performed between parental MCF-7, selected MCF-7-14, and aggressive mesenchymal MDA-MB-231 cells. Furthermore, using microarray expression profiles of these cells, we selected differentially expressed genes for their invasive potential, and performed pathway and network analysis to identify a set of interesting genes, which were evaluated by RT-PCR, flow cytometry or function-blocking antibody treatment.
MCF-7-14 cells had enhanced migratory and invasive ability compared with MCF-7 cells. Although MCF-7-14 cells, similar to MCF-7 cells, expressed E-cadherin but neither vimentin nor fibronectin, beta-catenin was expressed not only on the cell membrane but also in the nucleus. Furthermore, using gene expression profiles of MCF-7, MCF-7-14 and MDA-MB-231 cells, we demonstrated that MCF-7-14 cells have alterations in signaling pathways regulating cell migration and identified a set of genes (PIK3R1, SOCS2, BMP7, CD44 and CD24). Interestingly, MCF-7-14 and its invasive clone CL6 cells displayed increased CD44 expression and downregulated CD24 expression compared with MCF-7 cells. Anti-CD44 antibody treatment significantly decreased cell migration and invasion in both MCF-7-14 and MCF-7-14 CL6 cells as well as MDA-MB-231 cells.
MCF-7-14 cells are a novel model for breast cancer metastasis without requiring constitutive EMT and are categorized as a "metastable phenotype", which can be distinguished from both epithelial and mesenchymal cells. The alterations and characteristics of MCF-7-14 cells, especially nuclear beta-catenin and CD44 upregulation, may characterize invasive cell populations in breast cancer.
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The emu (
Dromaius novaehollandiae
) is a useful poultry animal farmed for fat, meat, and eggs. Genetic structure and relationships among farmed emu populations in Japan are unknown and the number of ...microsatellite markers for genetic analysis of the emu is insufficient. In this study, we isolated 16 microsatellites from the emu genome and developed ten new microsatellite markers. These microsatellite markers were used to characterize three farm emu populations in Japan. The number of alleles ranged from 3 to 13 and the expected (
H
E
) and observed heterozygosity (
H
O
) of these microsatellite loci was 0.187–0.802 and 0.179–0.647, respectively. The polymorphic information content ranged from 0.176 to 0.786. Positive inbreeding coefficient (
F
IS
) values were detected in all tested populations, and they ranged from 0.027 to 0.540. These results suggest that farm populations of the emu in Japan resulted from inbreeding. The fixation index (
F
ST
) values ranged from 0.026 to 0.061, and phylogenetic trees and population structure analysis confirmed no definitive genetic differentiation among the three populations. Therefore, these populations are at a relatively low level of genetic differentiation at present. The microsatellite markers developed in our study can be utilized for genetic analysis and preservation of genetic resources in the emu.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
An unconventional myosin encoded by the myosin VI gene (MYO6) contributes to hearing loss in humans. Homozygous mutations of MYO6 result in nonsyndromic profound congenital hearing loss, DFNB37. ...Kumamoto shaker/waltzer (ksv) mice harbor spontaneous mutations, and homozygous mutants exhibit congenital defects in balance and hearing caused by fusion of the stereocilia. We identified a Myo6c.1381G>A mutation that was found to be a p.E461K mutation leading to alternative splicing errors in Myo6 mRNA in ksv mutants. An analysis of the mRNA and protein expression in animals harboring this mutation suggested that most of the abnormal alternatively spliced isoforms of MYO6 are degraded in ksv mice. In the hair cells of ksv/ksv homozygotes, the MYO6 protein levels were significantly decreased in the cytoplasm, including in the cuticular plates. MYO6 and stereociliary taper-specific proteins were mislocalized along the entire length of the stereocilia of ksv/ksv mice, thus suggesting that MYO6 attached to taper-specific proteins at the stereociliary base. Histological analysis of the cochlear hair cells showed that the stereociliary fusion in the ksv/ksv mutants, developed through fusion between stereociliary bundles, raised cuticular plate membranes in the cochlear hair cells and resulted in incorporation of the bundles into the sheaths of the cuticular plates. Interestingly, the expression of the stereociliary rootlet-specific TRIO and F-actin binding protein (TRIOBP) was altered in ksv/ksv mice. The abnormal expression of TRIOBP suggested that the rootlets in the hair cells of ksv/ksv mice had excessive growth. Hence, these data indicated that decreased MYO6 levels in ksv/ksv mutants disrupt actin networks in the apical region of hair cells, thereby maintaining the normal structure of the cuticular plates and rootlets, and additionally provided a cellular basis for stereociliary fusion in Myo6 mutants.
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