Taken together, these results indicated that the fluorescent reporter genes were inserted into loci without disrupting or disturbing endogenous genes. Open in a separate window Fig. and were immunohistochemically demonstrated to match their respective germ layer-specific marker protein at E7.5. Taken together, these observations suggest that the knockin reporter mice may be useful for comprehensive analysis of gene function in germ layer formation. gene [11]. In this driver mouse strain, the function of insulin was maintained and the gene was expressed under the transcriptional regulation of because the gene including 2A sequences derived from porcine teschovirus-1 (P2A) was integrated at a site before the gene stop codon. Thus, the bicistronic knockin system is generally helpful for production of a reporter mouse strain that maintains the intact function of the respective target gene. To generate fluorescent proteins in each germ layer of the mouse gastrula, we used three genes, orthodenticle homeobox 2 (encoded Brachyury, and SRY (sex determining region Y)-box 17 (gene Igf1 is expressed first from the primitive streak during gastrulation [14, 18]. Lastly, establishment of the definitive endoderm is dependent on the activities of transcription factors, including members of the Sox and forkhead domain families. Especially, SOX17 plays an important role in endoderm development [15]. Therefore, in the present study, we used CRISPR/Cas9-mediated genome editing in mouse zygotes to generate bicistronic reporter knockin mouse strains, to allow visual identification of the endodermal, ectodermal, and mesodermal tissues, respectively, during gastrulation. Materials and Methods Animals C57BL/6J mice were purchased from Charles River Laboratories (Yokohama, Japan). Mice were maintained in plastic cages under pathogen-free conditions in a room maintained at 23.5 2.5C and 52.5 12.5% relative humidity under a EC089 14-h light:10-h dark cycle. Mice had free access to EC089 commercial chow (MF; Oriental Yeast Co., Ltd., Tokyo, Japan) and filtered water. Animal experiments were carried out in a humane manner with approval from the Institutional Animal Experiment Committee EC089 of the University of Tsukuba in accordance with the Regulations for Animal Experiments of the University of Tsukuba and Fundamental Guidelines for Proper Conduct of EC089 Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Cell culture and transfection HEK293T cells were cultured in Dulbeccos modified Eagles medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, in an atmosphere of 5% CO2 at 37C. Briefly, 3.5 104 HEK293T cells were transfected with and vectors of the target genes by Lipofectamine LTX (Thermo Fisher Scientific, Wilmington, DE, USA) or 6 105 HEK293T cells were transfected with by polyethylenimine (Sigma-Aldrich, St. Louis, MO, USA). After 24 h, the HEK293T cells were observed by fluorescence microscopy (BZ-X710; Keyence, Osaka, Japan). Vector construction CRISPR oligo DNAs (Supplementary Table 1) were annealed, purified, and inserted into the vector (Addgene plasmid 42230, a gift from Dr. Feng Zhang [5]) to yield constructs designated as genes containing the CRISPR target were amplified with the primers listed in Supplementary Table 2 and inserted into the vector to produce were amplified (Supplementary Table 3) and the PCR products were cloned into the reporter platform plasmids (Sox17 donor plasmid), were then amplified with PrimeSTAR? GXL DNA Polymerase (Takara, Otsu, Japan) (Supplementary Table 4) and cloned into pCX-V5-HA platform plasmids. The resultant plasmids were designated as vector and 10 ng/genes were fused with and used as reporters for the endoderm, ectoderm, and mesoderm, respectively. Moreover, to express.