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.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1400-x) contains supplementary material, which is available to authorized users.

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 wel 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.

BackgroundIn 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?MethodsThroughout 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.ResultsMCF-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, β-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.ConclusionsMCF-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 β-catenin and CD44 upregulation, may characterize invasive cell populations in breast cancer.

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.

Hair length in mammals is generally regulated by the hair cycle, and its disruption leads to abnormal hair morphogenesis in several species. FGF5, one of the hair cycle regulators, has a role in inducing catagen, and that mutation causes abnormal hair length in both sexes in humans, mice, dogs, and cats. Male-dominant long-haired coat (MALC) is an inbred strain of Syrian hamster exhibiting spontaneous long hair in males. After castration, MALC exhibited significantly shorter hair than the control individuals, but testosterone administration to castrated MALC showed reversion to the original phenotype. Moreover, flutamide administration led to MALC phenotype repression. Histological analysis revealed that hair follicle regression was shown in the wild-type 4 weeks after depilation, but that of MALC remained in the anagen phase. We detected a c.546delG of Fgf5 in MALC (Fgf5malc) that might lead to truncation resulting from a frame shift in FGF5 (p.Arg184GlyfsX6). Additionally, homozygous Fgf5malc was only detected in long-haired (Slc:Syrian×MALC)F2 and (J-2-Nn×MALC)F2 progenies, and all homozygous wild and heterozygous Fgf5malc individuals showed normal hair length. Thus, Fgf5malc leads to male-dominant long hair via a prolonged anagen phase which is affected by testosterone in hamsters. To our knowledge, this report is the first to present the sexual dimorphism of hair length caused by the Fgf5 mutation.