Search Results (10)

Aralia elata is an important herb due to the abundance of pentacyclic triterpenoid saponins whose important precursors are squalene and OA. Here, we found that MeJA treatment promoted both precursors accumulation, especially the latter, in transgenic A. elata, overexpressing a squalene synthase gene from Panax notoginseng(PnSS). In this study, Rhizobium-mediated transformation was used to express the PnSS gene. Gene expression analysis and high-performance liquid chromatography (HPLC) were used to identify the effect of MeJA on squalene and OA accumulation. The PnSS gene was isolated and expressed in A. elata. Transgenic lines showed a very high expression of the PnSS gene and farnesyl diphosphate synthase gene (AeFPS) and a slightly higher squalene content than the wild-type, but endogenous squalene synthase (AeSS), squalene epoxidase (AeSE), and β-amyrin synthase (Aeβ-AS) gene were decreased as well as OA content. Following one day of MeJA treatment, the expression levels of PeSS, AeSS, and AeSE genes increased significantly. On day 3, the maximum content of both products reached 17.34 and 0.70 mg·g^−1, which increased 1.39- and 4.90-fold than in the same lines without treatment. Transgenic lines expressing PnSS gene had a limited capability to promote squalene and OA accumulation. MeJA strongly activated their biosynthesis pathways, leading to enhance yield. Full article

Clustered regularly interspaced short palindromic repeats (CRISPR) and the associated protein (Cas) gene editing can induce P53 activation, large genome fragment deletions, and chromosomal structural variations. Here, gene expression was detected in host cells using transcriptome sequencing following CRISPR/Cas9 gene editing. We found that the gene editing reshaped the gene expression, and the number of differentially expressed genes was correlated with the gene editing efficiency. Moreover, we found that alternative splicing occurred at random sites and that targeting a single site for gene editing may not result in the formation of fusion genes. Further, gene ontology and KEGG enrichment analysis showed that gene editing altered the fundamental biological processes and pathways associated with diseases. Finally, we found that cell growth was not affected; however, the DNA damage response protein—γH2AX—was activated. This study revealed that CRISPR/Cas9 gene editing may induce cancer-related changes and provided basic data for research on the safety risks associated with the use of the CRISPR/Cas9 system. Full article

Salt is an important environmental stress factor, which seriously affects the growth, development and distribution of plants. Chlorophyllase plays an important role in stress response. Nevertheless, little is known about the physiological and molecular mechanism of chlorophyll (Chlase, CLH) genes in plants. We cloned PeCLH2 from Populus euphratica and found that PeCLH2 was differentially expressed in different tissues, especially in the leaves of P. euphratica. To further study the role of PeCLH2 in salt tolerance, PeCLH2 overexpression and RNA interference transgenic lines were established in Populus alba × Populus glandulosa and used for salt stress treatment and physiologic indexes studies. Overexpressing lines significantly improved tolerance to salt treatment and reduced reactive oxygen species production. RNA interference lines showed the opposite. Transcriptome analysis was performed on leaves of control and transgenic lines under normal growth conditions and salt stress to predict genes regulated during salt stress. This provides a basis for elucidating the molecular regulation mechanism of PeCLH2 in response to salt stress and improving the tolerance of poplar under salt stress. Full article

The French mouse clinic (Institut Clinique de la Souris; ICS) has produced more than 2000 targeting vectors for ‘à la carte’ mutagenesis in C57BL/6N mice. Although most of the vectors were used successfully for homologous recombination in murine embryonic stem cells (ESCs), a few have failed to target a specific locus after several attempts. We show here that co-electroporation of a CRISPR plasmid with the same targeting construct as the one that failed previously allows the systematic achievement of positive clones. A careful validation of these clones is, however, necessary as a significant number of clones (but not all) show a concatemerization of the targeting plasmid at the locus. A detailed Southern blot analysis permitted characterization of the nature of these events as standard long-range 5′ and 3′ PCRs were not able to distinguish between correct and incorrect alleles. We show that a simple and inexpensive PCR performed prior to ESC amplification allows detection and elimination of those clones with concatemers. Finally, although we only tested murine ESCs, our results highlight the risk of mis-validation of any genetically modified cell line (such as established lines, induced pluripotent stem cells or those used for ex vivo gene therapy) that combines the use of CRISPR/Cas9 and a circular double-stranded donor. We strongly advise the CRISPR community to perform a Southern blot with internal probes when using CRISPR to enhance homologous recombination in any cell type, including fertilized oocytes. Full article

The purpose of the study is to demonstrate coherent optical tomography and electroretinography techniques adopted from the human clinical practice to assess the morphology and function of the mouse retina in a high-throughput phenotyping environment. We present the normal range of wild-type C57Bl/6NCrl retinal parameters in six age groups between 10 and 100 weeks as well as examples of mild and severe pathologies resulting from knocking out a single protein-coding gene. We also show example data obtained by more detailed analysis or additional methods useful in eye research, for example, the angiography of a superficial and deep vascular complex. We discuss the feasibility of these techniques in conditions demanding a high-throughput approach such as the systemic phenotyping carried out by the International Mouse Phenotyping Consortium. Full article

Maize is one of the most important food crops, and maize kernel is one of the important components of maize yield. Studies have shown that the rice grain-size affecting gene GS5 increases the thousand-kernel weight by positively regulating the rice grain width and grain grouting rate. In this study, based on the GS5 transgenic maize obtained through transgenic technology with specific expression in the endosperm, molecular assays were performed on the transformed plants. Southern blotting results showed that the GS5 gene was integrated into the maize genome in a low copy number, and RT-PCR analysis showed that the exogenous GS5 gene was normally and highly expressed in maize. The agronomic traits of two successive generations showed that certain lines were significantly improved in yield-related traits, and the most significant changes were observed in the OE-34 line, where the kernel width increased significantly by 8.99% and 10.96%, the 100-kernel weight increased by 14.10% and 10.82%, and the ear weight increased by 13.96% and 15.71%, respectively; however, no significant differences were observed in the plant height, ear height, kernel length, kernel row number, or kernel number. In addition, the overexpression of the GS5 gene increased the grain grouting rate and affected starch synthesis in the rice grains. The kernels’ starch content in OE-25, OE-34, and OE-57 increased by 10.30%, 7.39%, and 6.39%, respectively. Scanning electron microscopy was performed to observe changes in the starch granule size, and the starch granule diameter of the transgenic line(s) was significantly reduced. RT-PCR was performed to detect the expression levels of related genes in starch synthesis, and the expression of these genes was generally upregulated. It was speculated that the exogenous GS5 gene changed the size of the starch granules by regulating the expression of related genes in the starch synthesis pathway, thus increasing the starch content. The trans-GS5 gene was able to be stably expressed in the hybrids with the genetic backgrounds of the four materials, with significant increases in the kernel width, 100-kernel weight, and ear weight. In this study, the maize kernel size was significantly increased through the endosperm-specific expression of the rice GS5 gene, and good material for the functional analysis of the GS5 gene was created, which was of great importance in theory and application. Full article

Background: Insulin-like growth factor 2 is a growth-promoting factor that plays an important role in the growth and development of mammals. A nucleotide substitution in intron 3 of IGF2—which disrupts the ZBED6-binding site—affects muscle mass, organ size, and fat deposition in pigs. The ZBED6-binding site is also conserved in cattle. Methods: In the present study, we introduced mutations in the ZBED6-binding site in intron3 of IGF2 in bovine fetal fibroblasts using the CRISPR/Cas9 system, and investigated the effect of disruption of ZBED6 binding on IGF2 expression. Results: Eleven biallelic-mutant single-cell clones were established, three of which contained no foreign DNA residues. Single-cell clones 93 and 135 were used to produce cloned embryos. Dual-luciferase reporter assay in C2C12 cells demonstrated that the mutation in the ZBED6-binding site increases the promoter 3 activity of bovine IGF2. A total of 49 mutant cloned embryos were transplanted into surrogate cows. Unfortunately, all cloned embryos died before birth. IGF2 was found to be hypomethylated in the only fetus born (stillborn), which may have been due to the incomplete reprogramming. Conclusions: We efficiently constructed IGF2-edited cell lines and cloned embryos, which provided a theoretical basis and experimental materials for beef cattle breeding. Full article

As the basis of animal reproductive activity, normal spermatogenesis directly determines the efficiency of livestock production. An in-depth understanding of spermatogenesis will greatly facilitate animal breeding efforts and male infertility treatment. With the continuous development and application of gene editing technologies, they have become valuable tools to study the mechanism of spermatogenesis. Gene editing technologies have provided us with a better understanding of the functions and potential mechanisms of action of factors that regulate spermatogenesis. This review summarizes the applications of gene editing technologies, especially CRISPR/Cas9, in deepening our understanding of the function of spermatogenesis-related genes and disease treatment. The problems of gene editing technologies in the field of spermatogenesis research are also discussed. Full article

Animal cloning is of great importance to the production of transgenic and genome-edited livestock. Especially for multiple gene-editing operations, recloning is one of the most feasible methods for livestock. In addition, a multiple-round cloning method is practically necessary for animal molecular breeding. However, cloning efficiency remains extremely low, especially for serial cloning, which seriously impedes the development of livestock breeding based on genome editing technology. The incomplete reprogramming and failure in oocyte activation of some pluripotent factors were deemed to be the main reason for the low efficiency of animal recloning. Here, to overcome this issue, which occurred frequently in the process of animal recloning, we established a reporter system in which fluorescent proteins were driven by pig OCT4 or SOX2 promoter to monitor the reprogramming process in cloned and recloned pig embryos. We studied the effect of different histone deacetylase (HDAC) inhibitors on incomplete reprogramming. Our results showed that Trichostatin A (TSA) could activate pluripotent factors and significantly enhance the development competence of recloned pig embryos, while the other two inhibitors, valproic acid (VPA) and Scriptaid, had little effect on that. Furthermore, we found no difference in OCT4 mRNA abundance between TSA-treated and untreated embryos. These findings suggest that TSA remarkably improves the reprogramming state of pig recloned embryos by restoring the expression of incompletely activated pluripotent genes OCT4 and SOX2. Full article

Generation of transgenic organisms by pronuclear microinjection has become a routine procedure. However, while the process of DNA integration in the genome is well understood, we still do not know much about the recombination between transgene molecules that happens in the first moments after DNA injection. Most of the time, injected molecules are joined together in head-to-tail tandem repeats—the so-called concatemers. In this review, we focused on the possible concatenation mechanisms and how they could be studied with genetic reporters tracking individual copies in concatemers. We also discuss various features of concatemers, including palindromic junctions and repeat-induced gene silencing (RIGS). Finally, we speculate how cooperation of DNA repair pathways creates a multicopy concatenated insert. Full article