Additional file 1: of COP9 signalosome subunit 5A affects phenylpropanoid metabolism, trichome formation and transcription of key genes of a regulatory tri-protein complex in Arabidopsis

Table S1. Primers used in this study. (XLSX 10 kb

Additional file 3: of COP9 signalosome subunit 5A affects phenylpropanoid metabolism, trichome formation and transcription of key genes of a regulatory tri-protein complex in Arabidopsis

Table S2. Down-regulated gene expression more than 2-fold in sk372 compared to wild-type control. (XLSX 25 kb

Additional file 5: of COP9 signalosome subunit 5A affects phenylpropanoid metabolism, trichome formation and transcription of key genes of a regulatory tri-protein complex in Arabidopsis

Figure S2. Molecular characterization of sk372. (A) Southern blot; (B) homozygous determination of sk372. (D) CSN5a truncation analysis using cDNA as a template. Lanes 1 and 2, full length (938 bp) of CSN5a in WT and sk372, respectively; Lanes 3 and 4, amplified fragment from 162 bp to 886 bp in WT and sk372; Lanes 5 and 6, amplified fragment starting from bp 1034 to 3′ end in WT and sk372.The presence of transgene GL3 in the different independent lines of transgenic sk372. M, 1 kb plus DNA marker (Invitrogen); T1-T8, different independent lines of transgenic plants; WT, wild type control. (TIF 7690 kb

Additional file 6: of COP9 signalosome subunit 5A affects phenylpropanoid metabolism, trichome formation and transcription of key genes of a regulatory tri-protein complex in Arabidopsis

Figure S3. The presence of transgene GL3 in the different independent lines of 563 transgenic sk372. M, 1 kb plus DNA marker (Invitrogen); T1-T8, different independent lines of 564 transgenic plants; WT, wild type control. (TIF 879 kb

Additional file 2: of COP9 signalosome subunit 5A affects phenylpropanoid metabolism, trichome formation and transcription of key genes of a regulatory tri-protein complex in Arabidopsis

Figure S1. Overrepresented categories of enriched gene ontology based on differential gene expression between sk372 seedlings compared with wild type Arabidopsis. Circle sizes represent larger or smaller numbers of differentially expressed genes. Circle colours represent p values. (TIF 1781 kb

Additional file 4: of Transcriptomic and phytochemical analysis of the biosynthesis of characteristic constituents in tea (Camellia sinensis) compared with oil tea (Camellia oleifera)

Information about the genes involved in the flavonoid, theanine and caffeine biosynthesis pathways. Unigenes involved in three metabolic pathways were annotated by alignment to the Swiss-Prot, COG and KEGG databases with e-values less than 1 × 10−30. (XLSX 82 kb

Generation of Knock-In Pigs Carrying Oct4-tdTomato Reporter through CRISPR/Cas9-Mediated Genome Engineering

<div><p>The porcine pluripotent cells that can generate germline chimeras have not been developed. The Oct4 promoter-based fluorescent reporter system, which can be used to monitor pluripotency, is an important tool to generate authentic porcine pluripotent cells. In this study, we established a porcine Oct4 reporter system, wherein the endogenous Oct4 promoter directly controls red fluorescent protein (RFP). 2A-tdTomato sequence was inserted to replace the stop codon of the porcine Oct4 gene by homogenous recombination (HR). Thus, the fluorescence can accurately show the activation of endogenous Oct4. Porcine fetal fibroblast (PFF) lines with knock-in (KI) of the tdTomato gene in the downstream of endogenous Oct4 promoter were achieved using the CRISPR/CAS9 system. Transgenic PFFs were used as donor cells for somatic cell nuclear transfer (SCNT). Strong RFP expression was detected in the blastocysts and genital ridges of SCNT fetuses but not in other tissues. Two viable transgenic piglets were also produced by SCNT. Reprogramming of fibroblasts from the fetuses and piglets by another round of SCNT resulted in tdTomato reactivation in reconstructed blastocysts. Result indicated that a KI porcine reporter system to monitor the pluripotent status of cells was successfully developed.</p></div

Generation of an endogenous porcine Oct4-2A–tdTomato reporter.

<p>(A) Schematic of the method in generating porcine Oct4-2A–tdTomato KI allele. The genes Cas9 and sgOct4 were constructed in a single vector. The homologous arms of the donor vector are indicated as left arm (950 bp) and right arm (1350 bp). PCR primers (P1, P2 and P3) used for PCR genotyping are indicated by blue and red arrowheads. (B) Red fluorescence in porcine Oct4-2A–tdTomato reconstructed blastocysts derived from SCNT embryos. The porcine fibroblast colonies C51, C95, and C98 were reprogrammed to become highly efficient tdTomato-expressing blastocysts. Scale bar, 100 μm. (C) PCR genotyping using the primers indicated in the diagram detected the KI colones and embryos. PCR revealed wild type band (0 bp) and the 2A-tdTomato KI band (1000 bp) using primers P1/P2. PCR revealed the WT band (2300 bp) and the 2A-tdTomato KI band (3900 bp) using primers P1/P3. (D) The targeted porcine blastocysts remained tdTomato positive in the ICM (arrow), whereas disappeared in trophoblast cells (arrowhead), after 5 days cultured in ES medium. Scale bar, 50 μm. (E) The targeted porcine blastocysts differentiated into tdTomato negative colonies after being cultured for 15 days. Scale bar, 50 μm. (F) Q-PCR compared the endogenous Oct4 mRNA levels of porcine gene-target blastocysts cultured for 5 and 15 days. PFFs were used as negative control and porcine blastocysts without being cultured were used as positive control.</p

Analysis of the KI fetuses.

<p>(A) PCR genotyping detected the KI fetuses by using primers indicated in the diagram. PCR revealed wild type band (0 bp) and the 2A-tdTomato KI band (1000 bp) using primers P1/P2. PCR revealed the WT band (2300 bp) and the 2A-tdTomato KI band (3900 bp) using primers P1/P3. (B) TdTomato was expressed, with varying degree of fluorescence, in PGCs of genital ridges isolated from fetuses. (C) TdTomato was expressed in the genital ridges of target fetuses, but not in other tissues, such as intestines. tdTomato was not expressed in WT genital ridges. Bright field image of the tissue is shown on the lower right corner of each image. Scale bar, 1 mm. (D) PFFs cultured in vitro did not express tdTomato. Scale bar, 100 μm. (E) tdTomato was expressed in blastocysts after another round of SCNT using fibroblasts isolated from pOct4-2A-tdtoamto KI fetuses as nuclear donors. WT blastocysts did not express tdTomato. Scale bar, 100 μm. (F) Dome-like colonies formed 10 days after transfection did not express tdTomato. Scale bar, 50 μm. (G) RT-PCR was used to identify the transcription of endogenous and exogenous Oct4 in piPS-LCs. All colonies expressed exogenous Oct4 but not endogenous Oct4.</p