• BMB Reports
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• 제57권1호
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• pp.50-59
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• 2024

The application of gene engineering in livestock is necessary for various reasons, such as increasing productivity and producing disease resistance and biomedicine models. Overall, gene engineering provides benefits to the agricultural and research aspects, and humans. In particular, productivity can be increased by producing livestock with enhanced growth and improved feed conversion efficiency. In addition, the application of the disease resistance models prevents the spread of infectious diseases, which reduces the need for treatment, such as the use of antibiotics; consequently, it promotes the overall health of the herd and reduces unexpected economic losses. The application of biomedicine could be a valuable tool for understanding specific livestock diseases and improving human welfare through the development and testing of new vaccines, research on human physiology, such as human metabolism or immune response, and research and development of xenotransplantation models. Gene engineering technology has been evolving, from random, time-consuming, and laborious methods to specific, time-saving, convenient, and stable methods. This paper reviews the overall trend of genetic engineering technologies development and their application for efficient production of genetically engineered livestock, and provides examples of technologies approved by the United States (US) Food and Drug Administration (FDA) for application in humans.

• Journal of Microbiology and Biotechnology
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• 제27권10호
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• pp.1855-1866
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• 2017

The synergistic activation mediator (SAM) system can robustly activate endogenous gene expression by a single-guide RNA. This transcriptional modulation has been shown to enhance gene promoter activity and leads to epigenetic changes. Human $interferon-{\gamma}$ is a common natural glycoprotein involved in antiviral effects and inhibition of cancer cell growth. Large quantities of high-purity $interferon-{\gamma}$ are important for medical research and clinical therapy. To investigate the possibility of employing the SAM system to enhance endogenous human $interferon-{\gamma}$ with normal function in innate immunity, we designed 10 single-guide RNAs that target 200 bp upstream of the transcription start sites of the $interferon-{\gamma}$ genome, which could significantly activate the $interferon-{\gamma}$ promoter reporter. We confirmed that the system can effectively and highly activate $interferon-{\gamma}$ expression in several humanized cell lines. Moreover, we found that the $interferon-{\gamma}$ induced by the SAM system could inhibit tumorigenesis. Taken together, our results reveal that the SAM system can modulate epigenetic traits of non-immune cells through activating $interferon-{\gamma}$ expression and triggering JAK-STAT signaling pathways. Thus, this strategy could offer a novel approach to inhibit tumorigenesis without using exogenous $interferon-{\gamma}$.

• 한국해양바이오학회지
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• 제11권2호
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• pp.52-61
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• 2019

Over the past few decades, microalgae-based biotechnology conjugated with innovative CRISPR/Cas9-mediated genetic engineering has been attracted much attention for the cost-effective and eco-friendly value-added compounds production. However, the discharge of reproducible living modified organism (LMO) into environmental condition potentially causes serious problem in aquatic environment, and thus it is essential to assess potential environmental risk for human health. Accordingly, in this study, we monitored discharged genetically modified microalgae (GMM) near the research complex which is located in Daejeon, South Korea. After testing samples obtained from 6 points of near streams, several green-colored microalgal colonies were detected under hygromicin-containing agar plate. By identification of selection marker genes, the GMM was not detected from all the samples. For the lab-scale environmental risk assessment of GMM, acute toxicity test using rotifer Brachionus calcyflorus was performed by feeding GMM. After feeding, there was no significant difference in mortality between WT and transformant Chlamydomonas reinhardtii. According to further analysis of horizontal transfer of green fluorescence protein (GFP)-coding gene after 24 h of incubation in synthetic freshwater, we concluded that the GFP-expressed gene not transferred into predator. However, further risk assessments and construction of standard methods including prolonged toxicity test are required for the accurate ecological risk assessment.

• The Korean Journal of Physiology and Pharmacology
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• 제24권3호
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• pp.233-240
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• 2020

Autophagy regulators are often effective as potential cancer therapeutic agents. Here, we investigated paclitaxel sensitivity in cells with knockout (KO) of ATG5 gene. The ATG5 KO in multidrug resistant v-Ha-ras-transformed NIH 3T3 cells (Ras-NIH 3T3/Mdr) was generated using the CRISPR/Cas9 technology. The qPCR and LC3 immunoblot confirmed knockout of the gene and protein of ATG5, respectively. The ATG5 KO restored the sensitivity of Ras-NIH 3T3/Mdr cells to paclitaxel. Interestingly, ATG5 overexpression restored autophagy function in ATG5 KO cells, but failed to rescue paclitaxel resistance. These results raise the possibility that low level of resistance to paclitaxel in ATG5 KO cells may be related to other roles of ATG5 independent of its function in autophagy. The ATG5 KO significantly induced a G₂/M arrest in cell cycle progression. Additionally, ATG5 KO caused necrosis of a high proportion of cells after paclitaxel treatment. These data suggest that the difference in sensitivity to paclitaxel between ATG5 KO and their parental MDR cells may result from the disparity in the proportions of necrotic cells in both populations. Thus, our results demonstrate that the ATG5 KO in paclitaxel resistant cells leads to a marked G₂/M arrest and sensitizes cells to paclitaxel-induced necrosis.

• The Korean Journal of Physiology and Pharmacology
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• 제22권4호
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• pp.457-465
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• 2018

The expression of BCL-2 interacting cell death suppressor (BIS), an anti-stress or anti-apoptotic protein, has been shown to be regulated at the transcriptional level by heat shock factor 1 (HSF1) upon various stresses. Recently, HSF1 was also shown to bind to BIS, but the significance of these protein-protein interactions on HSF1 activity has not been fully defined. In the present study, we observed that complete depletion of BIS using a CRISPR/Cas9 system in A549 non-small cell lung cancer did not affect the induction of heat shock protein (HSP) 70 and HSP27 mRNAs under various stress conditions such as heat shock, proteotoxic stress, and oxidative stress. The lack of a functional association of BIS with HSF1 activity was also demonstrated by transient downregulation of BIS by siRNA in A549 and U87 glioblastoma cells. Endogenous BIS mRNA levels were significantly suppressed in BIS knockout (KO) A549 cells compared to BIS wild type (WT) A549 cells at the constitutive and inducible levels. The promoter activities of BIS and HSP70 as well as the degradation rate of BIS mRNA were not influenced by depletion of BIS. In addition, the expression levels of the mutant BIS construct, in which 14 bp were deleted as in BIS-KO A549 cells, were not different from those of the WT BIS construct, indicating that mRNA stability was not the mechanism for autoregulation of BIS. Our results suggested that BIS was not required for HSF1 activity, but was required for its own expression, which involved an HSF1-independent pathway.

• Molecules and Cells
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• 제42권10호
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• pp.711-720
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• 2019

Sink strength optimizes sucrose import, which is fundamental to support developing seed grains and increase crop yields, including those of rice (Oryza sativa). In this regard, little is known about the function of vacuolar invertase (VIN) in controlling sink strength and thereby seed size. Here, in rice we analyzed mutants of two VINs, OsVIN1 and OsVIN2, to examine their role during seed development. In a phenotypic analysis of the T-DNA insertion mutants, only the OsVIN2 mutant osvin2-1 exhibited reduced seed size and grain weight. Scanning electron microscopy analysis revealed that the small seed grains of osvin2-1 can be attributed to a reduction in spikelet size. A significant decrease in VIN activity and hexose level in the osvin2-1 spikelets interfered with spikelet growth. In addition, significant reduction in starch and increase in sucrose, which are characteristic features of reduced turnover and flux of sucrose due to impaired sink strength, were evident in the pre-storage stage of osvin2-1 developing grains. In situ hybridization analysis found that expression of OsVIN2 was predominant in the endocarp of developing grains. A genetically complemented line with a native genomic clone of OsVIN2 rescued reduced VIN activity and seed size. Two additional mutants, osvin2-2 and osvin2-3 generated by the CRISPR/Cas9 method, exhibited phenotypes similar to those of osvin2-1 in spikelet and seed size, VIN activity, and sugar metabolites. These results clearly demonstrate an important role of OsVIN2 as sink strength modulator that is critical for the maintenance of sucrose flux into developing seed grains.

• 한국버섯학회지
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• 제19권3호
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• pp.256-259
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• 2021

현재 양송이 품종의 개발은 1980년대에 개발된 방법에 의존하여 진행되고 있다. 유전자가위를 이용한 유전자교정 기술이 다양한 분야에서 각광받고 있고, 이 기술을 버섯 육종에도 적용하기 위하여 진행된 이 연구에서는, CRISPR/Cas9 활용에 필수적인 원형질체 분리 효율을 1.0 × 10⁸/mL까지 안정적으로 끌어올렸고, spermidine을 이용하여 PEG 형질전환의 효율 또한 기존 방법에 비해 100배가량 끌어올렸음을 보고한다.

• Journal of Veterinary Science
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• 제22권3호
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• pp.36.1-36.12
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• 2021

Background: Mouse hepatitis virus (MHV) A59 is a highly infectious pathogen and starts in the respiratory tract and progresses to systemic infection in laboratory mice. The complement system is an important part of the host immune response to viral infection. It is not clear the role of the classical complement pathway in MHV infection. Objectives: The purpose of this study was to determine the importance of the classical pathway in coronavirus pathogenesis by comparing C1qa KO mice and wild-type mice. Methods: We generated a C1qa KO mouse using CRISPR/Cas9 technology and compared the susceptibility to MHV A59 infection between C1qa KO and wild-type mice. Histopathological and immunohistochemical changes, viral loads, and chemokine expressions in both mice were measured. Results: MHV A59-infected C1qa KO mice showed severe histopathological changes, such as hepatocellular necrosis and interstitial pneumonia, compared to MHV A59-infected wild-type mice. Virus copy numbers in the olfactory bulb, liver, and lungs of C1qa KO mice were significantly higher than those of wild-type mice. The increase in viral copy numbers in C1qa KO mice was consistent with the histopathologic changes in organs. These results indicate that C1qa deficiency enhances susceptibility to MHV A59 systemic infection in mice. In addition, this enhanced susceptibility effect is associated with dramatic elevations in spleen IFN-γ, MIP-1 α, and MCP-1 in C1qa KO mice. Conclusions: These data suggest that C1qa deficiency enhances susceptibility to MHV A59 systemic infection, and activation of the classical complement pathway may be important for protecting the host against MHV A59 infection.

• Journal of Microbiology and Biotechnology
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• 제31권4호
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• pp.570-583
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• 2021

Pyrococcus furiosus α-amylase can hydrolyze α-1,4 linkages in starch and related carbohydrates under hyperthermophilic condition (~ 100℃), showing great potential in a wide range of industrial applications, while its relatively low productivity from heterologous hosts has limited the industrial applications. Bacillus subtilis, a gram-positive bacterium, has been widely used in industrial production for its non-pathogenic and powerful secretory characteristics. This study was conducted to increase production of P. furiosus α-amylase in B. subtilis through three strategies. Initial experiments showed that co-expression of P. furiosus molecular chaperone peptidyl-prolyl cis-trans isomerase through genomic integration mode, using a CRISPR/Cas9 system, increased soluble amylase production. Therefore, considering that native P. furiosus α-amylase is produced within a hyperthermophilic environment and is highly thermostable, heat treatment of intact culture at 90℃ for 15 min was performed, thereby greatly increasing soluble amylase production. After optimization of the culture conditions (nitrogen source, carbon source, metal ion, temperature and pH), experiments in a 3-L fermenter yielded a soluble activity of 3,806.7 U/ml, which was 3.3- and 28.2-fold those of a control without heat treatment (1,155.1 U/ml) and an empty expression vector control (135.1 U/ml), respectively. This represents the highest P. furiosus α-amylase production reported to date and should promote innovation in the starch liquefaction process and related industrial productions. Meanwhile, heat treatment, which may promote folding of aggregated P. furiosus α-amylase into a soluble, active form through the transfer of kinetic energy, may be of general benefit when producing proteins from thermophilic archaea.

• The Plant Pathology Journal
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• 제37권6호
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• pp.555-565
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• 2021

Bacteriophages infecting Acidovorax citrulli, the causal agent of bacterial fruit blotch, have been proven to be effective for the prevention and control of this disease. However, the occurrence of bacteriophage-resistant bacteria is one of hurdles in phage biocontrol and the understanding of phage resistance in this bacterium is an essential step. In this study, we aim to investigate possible phage resistance of A. citrulli and relationship between phage resistance and pathogenicity, and to isolate and characterize the genes involved in these phenomena. A phage-resistant and less-virulent mutant named as AC-17-G1 was isolated among 3,264 A. citrulli Tn5 mutants through serial spot assays and plaque assays followed by pathogenicity test using seed coating method. The mutant has the integrated Tn5 in the middle of a cupin protein gene. This mutant recovered its pathogenicity and phage sensitivity by complementation with corresponding wild-type gene. Site-directed mutation of this gene from wild-type by CRISPR/Cas9 system resulted in the loss of pathogenicity and acquisition of phage resistance. The growth of AC-17-G1 in King's B medium was much less than the wild-type, but the growth turned into normal in the medium supplemented with D-mannose 6-phosphate or D-fructose 6-phosphate indicating the cupin protein functions as a phosphomannos isomerase. Sodium dodecyl sulfa analysis of lipopolysaccharide (LPS) extracted from the mutant was smaller than that from wild-type. All these data suggest that the cupin protein is a phosphomannos isomerase involved in LPS synthesis, and LPS is an important determinant of pathogenicity and phage susceptibility of A. citrulli.