scholarly journals A protocol to generate SPH-OminiCMV-Ents mESCs

2021 ◽  
Author(s):  
Ni Gao ◽  
Jing Hu ◽  
Haibo Zhou ◽  
Hui Yang
Keyword(s):  
Genetic Information ◽  
Cell Biology ◽  
Expression Level ◽  
Natural Context ◽  
Endogenous Promoter ◽  
Switch System ◽  
Endogenous Genes ◽  
Low Expression

Abstract Detection of genetic information in the natural context is essential to understand the biological principles. However, the ability to sense the activity of endogenous genes is limited for conventional tools. Here, we developed a highly programmable sgRNA switch system (Ents) that enables detection of endogenous genes and lncRNAs, even with very low expression level. This protocol is related to the publication “Endogenous promoter-driven sgRNA for monitoring the expression of low-abundant genes and lncRNAs” in Nature Cell Biology.

scholarly journals Development of a Transgenic Flammulina velutipes Oral Vaccine for Hepatitis B

2019 ◽  
Vol 57 (1) ◽  
pp. 105-112 ◽  
Author(s):  
Li-Hsin Huang ◽  
Hao-Yeh Lin ◽  
Ying-Tzu Lyu ◽  
Chiau-Ling Gung ◽  
Ching-Tsan Huang
Keyword(s):  
Hepatitis B ◽  
Oral Vaccine ◽  
Expression Level ◽  
Flammulina Velutipes ◽  
Total Soluble Protein ◽  
Control Group ◽  
Fruiting Bodies ◽  
Oral Vaccines ◽  
Virus Surface ◽  
Low Expression

Orally administered fungal vaccines show promise for the prevention of infectious diseases. Edible mushrooms are deemed appropriate hosts to produce oral vaccines due to their low production cost and low risk of gene contamination. However, their low expression level of antigens has limited the potential development of oral vaccines using mushrooms. The low expression level might result from impurity of the transgenic mycelia since dikaryotic mycelia are commonly used as transformation materials. In this study, stable transgenic hepatitis B virus surface antigen (HBsAg) in Flammulina velutipes transformants was obtained by Agrobacterium-mediated transformation, followed by fruiting and basidiospore mating. The formation of HBsAg was detected by western blot analysis. The expression levels of HBsAg in transgenic F. velutipes fruiting bodies were (129.3±15.1), (110.9±1.7) and (161.1±8.5) ng/g total soluble protein. However, the values may be underestimated due to incomplete protein extraction. Two of the four pigs in the experimental group produced positive anti-HBsAg-specific IgG after being fed the HBsAg transgenic F. velutipes fruiting bodies for 20 weeks, while no anti-HBsAg antibody was detected in the control group. One of the positive pigs had HBsAg titres of 5.36 and 14.9 mIU/mL in weeks 10 and 14, respectively, but expression faded thereafter. The other positive pig displayed HBsAg titres of 9.75, 17.86 and 39.87 mIU/mL in weeks 14, 18 and 20, respectively. The successful immunogenicity in pigs fed transgenic F. velutipes fruiting bodies demonstrated the potential of using the fungus as an oral vaccine.

scholarly journals Discovery of pluripotency-associated microRNAs in rabbit preimplantation embryos and embryonic stem-like cells

Reproduction ◽  
2013 ◽  
Vol 145 (4) ◽  
pp. 421-437 ◽  
Author(s):  
Pouneh Maraghechi ◽  
László Hiripi ◽  
Gábor Tóth ◽  
Babett Bontovics ◽  
Zsuzsanna Bősze ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Cell Biology ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Expression Level ◽  
Preimplantation Embryos ◽  
Regulatory Function ◽  
Low Level ◽  
Non Coding Rnas

MicroRNAs (miRNAs) are small non-coding RNAs that regulate multiple biological processes. Increasing experimental evidence implies an important regulatory role of miRNAs during embryonic development and in embryonic stem (ES) cell biology. In the current study, we have described and analyzed the expression profile of pluripotency-associated miRNAs in rabbit embryos and ES-like cells. The rabbit specific ocu-miR-302 and ocu-miR-290 clusters, and three homologs of the human C19MC cluster (ocu-miR-512, ocu-miR-520e, and ocu-miR-498) were identified in rabbit preimplantation embryos and ES-like cells. The ocu-miR-302 cluster was highly similar to its human homolog, while ocu-miR-290 revealed a low level of evolutionary conservation with its mouse homologous cluster. The expression of the ocu-miR-302 cluster began at the 3.5 days post-coitum early blastocyst stage and they stayed highly expressed in rabbit ES-like cells. In contrast, a high expression level of the ocu-miR-290 cluster was detected during preimplantation embryonic development, but a low level of expression was found in rabbit ES-like cells. Differential expression of the ocu-miR-302 cluster and ocu-miR-512 miRNA was detected in rabbit trophoblast and embryoblast. We also found that Lefty has two potential target sites in its 3′UTR for ocu-miR-302a and its expression level increased upon ocu-miR-302a inhibition. We suggest that the expression of the ocu-miR-302 cluster is characteristic of the rabbit ES-like cell, while the ocu-miR-290 cluster may play a crucial role during early embryonic development. This study presents the first identification, to our knowledge, of pluripotency-associated miRNAs in rabbit preimplantation embryos and ES-like cells, which can open up new avenues to investigate the regulatory function of ocu-miRNAs in embryonic development and stem cell biology.

scholarly journals A link between expression level of long-non-coding RNA ZFAS1 in breast tissue of healthy women and obesity

2018 ◽  
Vol 33 (4) ◽  
pp. 500-506 ◽  
Author(s):  
Yaser Mansoori ◽  
Mohammad Bagher Tabei ◽  
Alireza Askari ◽  
Pantea Izadi ◽  
Abdolreza Daraei ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Negative Correlation ◽  
Breast Tissue ◽  
Breast Cancer Incidence ◽  
Reproductive History ◽  
Expression Level ◽  
Expression Levels ◽  
Healthy Women ◽  
Low Expression ◽  
High Bmi

Background: Epidemiological and experimental literature indicates that the risk of breast cancer incidence is strongly linked to hormone-dependent factors, including reproductive history and obesity. However, the molecular mechanisms underlying the association between these factors and breast cancer risk are poorly understood. The aim of this study, therefore, was to determine whether obesity and reproductive history are associated with expression levels of two breast cancer-related long non-coding RNAs (lncRNAs), namely ZFAS1 and SRA1 in cancer-free breast tissues of women. Methods: In the current research, 145 healthy women were recruited, and the quantitative expression levels of the two lncRNAs were determined through qPCR assay after gathering the mammoplasty breast tissue samples. Results: It was found that women with body mass index (BMI)≥30 kg/m2 and BMI 25–29 kg/m2 show a low expression of ZFAS1 compared to the BMI<25 kg/m2 ( P=0.031 and P=0.027, respectively). Then, the correlation analysis disclosed a negative correlation of ZFAS1 low expression with increasing BMI (r=−0.194, P=0.019). Interestingly, this analysis demonstrated a negative correlation between low expression of the ZFAS1 and high BMI in women with menarche age below 14 (r=−221; P=0.028). Lastly, it was also revealed that there was a negative association of the low expression level of ZFAS1 with increasing BMI in women through regression models (B=−0.048, P=0.019). Conclusions: These findings suggest interesting clues about the links between high BMI and the expression levels of ZFAS1 in non-diseased breasts that may help us better understand the underlying mechanisms through which obesity contributes to breast carcinogenesis. However, such results need more validations in future research.

The effect of miRNAs and MALAT1 related with the prognosis of Her-2 positive breast cancer patients with lymph node metastasis

2020 ◽  
Author(s):  
Zhao Hongcan ◽  
Yang Hongjian ◽  
Zhang Xiping
Keyword(s):  
Lymph Node ◽  
Lymph Node Metastasis ◽  
Lymph Nodes ◽  
High Expression ◽  
Expression Level ◽  
Lymph Nodes Metastasis ◽  
Node Metastasis ◽  
Her 2 ◽  
Proliferation And Invasion ◽  
Low Expression

Abstract Background: To analyze and screen the miRNAs associated with lymph node metastasis of breast cancer (BC), and to explore the roles of these miRNAs in the proliferation, invasion and prognosis of BC. Methods: MicroRNAs associated with lymph node metastasis in Her-2 positive BC was screened by TCGA database. The qRT-PCR was used to verify theses 5 miRNAs in 30 cases of Her-2 positive BC with lymph node metastasis of different degree. The tumor tissue samples were divided into non-lymph node metastasis group, ≤ 3 lymph node metastasis group and > 3 lymph node metastasis group. In addition, 10 cases of paracancerous tissues were considered as paracancerous control group. Pearson correlation analysis was used to analysis the relationship of 5 miRNAs and MALAT1 with Her-2 positive BC patients' clinicopathological characteristics and prognosis. CCK8 and Transwell experiments were used to detect the effects of miR-143 and miR-455 on the proliferation and invasion of Her-2 positive BC cells (MDA-MB-453). Results: Five kinds of miRNA (miR-143, miR-196a, miR-455, miR-9 and miR-92a) related with Her-2 positive BC with lymph node metastasis were screened by TCGA database. The detecting results of qRT-PCR showed that the levels of miR-143, miR-196a, miR-9 and MALAT1 increased with the increased number of lymph nodes. The expression level of miR-143 in the group of ≤ 3 lymph nodes metastasis and > 3 lymph nodes metastasis was significantly higher than that in the group of non-lymph nodes metastasis (P<0.001), and that in the group of > 3 lymph nodes metastasis was significantly higher than that in the group of ≤ 3 lymph nodes metastasis (P<0.001). The expression level of miR-196a in the group of ≤ 3 lymph nodes metastasis and > 3 lymph nodes metastasis was significantly higher than that in the group of non-lymph nodes metastasis (P<0.001), and that in the group of > 3 lymph nodes metastasis was significantly higher than that in the group of ≤ 3 lymph nodes metastasis (P<0.001). The expression level of miR-455 in the group of ≤ 3 lymph nodes metastasis and > 3 lymph nodes metastasis was significantly lower than that in the group of non-lymph nodes metastasis (P<0.001), and that in the group of > 3 lymph nodes metastasis was significantly lower than that in the group of ≤ 3 lymph nodes metastasis (P<0.001). The expression level of MALAT1 in the group of ≤ 3 lymph nodes metastasis and > 3 lymph nodes metastasis was significantly higher than that in the group of non-lymph nodes metastasis (P<0.001), and that in the group of > 3 lymph nodes metastasis was significantly higher than that in the group of ≤ 3 lymph nodes metastasis (P<0.01). Pearson correlation analysis showed that the expression levels of miR-455-5p, miR-196a-5p and MALAT1 were negatively correlated, positively correlated and positively correlated with the pathological stages of Her-2 positive BC, respectively. The results of survival analysis showed that RFS of patients with high expression of miR-196a, miR-92a and MALAT1 was significantly lower than that of patients with low expression (P<0.05), and OS and RFS of patients with high expression of miR-9 were significantly lower than those of patients with low expression, while OS and RFS of patients with high expression of miR-455 were significantly higher than those of patients with low expression (P<0.05). Cytological experiments showed that up regulation of miR-455 significantly inhibited the proliferation and invasion of BC cells, while down regulation of miR-143 significantly inhibited the proliferation and invasion of BC cells and the expression of MALAT1 (P<0.05). Conclusion: High expression of miR-143, miR-9, miR-196a, MALAT1 and low expression of miR-455 are related to the degree of lymph node metastasis of Her-2-positive BC patients, indicating poor prognosis. Down-regulation of miR-455 and up-regulation of miR-143 and MALAT1 can promote the cell proliferation and invasion of Her-2-positive BC.

scholarly journals Low expression level but potent antigen presenting function of CD1d on monocyte lineage cells

2000 ◽  
Vol 30 (12) ◽  
pp. 3468-3477 ◽  
Author(s):  
Franca M. Spada ◽  
Frank Borriello ◽  
Masahiko Sugita ◽  
Gerald F. M. Watts ◽  
Yasuhiko Koezuka ◽  
...  
Keyword(s):  
Expression Level ◽  
Antigen Presenting ◽  
Low Expression

Action of SDF-1/CXCR4 axis in liver metastasis of colorectal cancer.

2012 ◽  
Vol 30 (4_suppl) ◽  
pp. 530-530 ◽  
Author(s):  
Shinichiro Yamada ◽  
Mitsuo Shimada ◽  
Toru Utsunomiya ◽  
Satoru Imura ◽  
Yuji Morine ◽  
...  
Keyword(s):  
Liver Metastasis ◽  
Colorectal Liver Metastasis ◽  
Normal Liver ◽  
High Expression ◽  
Expression Level ◽  
Expression Levels ◽  
High Expression Group ◽  
Cxcr4 Axis ◽  
Low Expression ◽  
Liver Tissues

530 Background: It has recently been suggested that the SDF-1/CXCR4 axis is involved in tumor progression, angiogenesis, metastasis, and survival in various cancer. In this study, we investigate the possible role of SDF-1/CXCR4 axis in colorectal liver metastasis. Methods: Both primary colorectal tumors and liver metastatic tumors were obtained from 12 patients with colorectal liver metastasis. Expression levels of CXCR4 and SDF-1 were determined using RT-PCR. In 4 patients with benign liver disease, the expression level of SDF-1 in normal liver tissues was also determined. We divided the 12 patients into two groups; high expression group (n=6) and low expression group (n=6) according to each expression level of SDF-1 and CXCR4, and compared the clinicopathological factors between the two groups. Results: 1. CXCR4 expression levels in primary tumor: The frequency of the peritoneal dissemination in the CXCR4 high expression group was higher than in the low expression group (p=0.07). Moreover, overall survival rate in the CXCR4 high expression group was significantly lower than that in the low expression group (3 year-survival rate: 67% vs. 100%, p<0.05). 2. CXCR4 in metastatic tumor tissues and SDF-1 in non-tumor liver tissues: The expression level of SDF-1 in non-tumor liver tissues was significantly higher than that in normal liver tissues (p<0.01). A significant correlation between the CXCR4 expression levels in metastatic tumor tissues and SDF-1 expression levels of non-tumor liver tissues (p<0.05). The number of metastatic liver tumors in the SDF-1 high expression group tended to be larger than that in the low expression group (p=0.12). Conclusions: The present data suggest that there is a significant association of the SDF-1/CXCR4 axis with enhanced liver metastasis and poor prognosis of the patients with colorectal liver metastasis. Furthermore, an enhanced expression of SDF-1 in non-tumor liver tissues may have an important role in the formation of liver metastasis.

scholarly journals Optogenetic regulation of endogenous gene transcription in mammals

2019 ◽  
Vol 23 (2) ◽  
pp. 219-225
Author(s):  
E. S. Omelina ◽  
A. V. Pindyurin
Keyword(s):  
Cell Biology ◽  
Near Infrared ◽  
Rapid Development ◽  
Infrared Light ◽  
Transcription Activator ◽  
Endogenous Gene ◽  
Advantages And Disadvantages ◽  
Ideal System ◽  
Endogenous Genes ◽  
Inducible Systems

Despite the rapid development of approaches aimed to precisely control transcription of exogenous genes in time and space, design of systems providing similar tight regulation of endogenous gene expression is much more challenging. However, finding ways to control the activity of endogenous genes is absolutely necessary for further progress in safe and effective gene therapies and regenerative medicine. In addition, such systems are of particular interest for genetics, molecular and cell biology. An ideal system should ensure tunable and reversible spatio-temporal control over transcriptional activity of a gene of interest. Although there are drug-inducible systems for transcriptional regulation of endogenous genes, optogenetic approaches seem to be the most promising for the gene therapy applications, as they are noninvasive and do not exhibit toxicity in comparison with druginducible systems. Moreover, they are not dependent on chemical inducer diffusion rate or pharmacokinetics and exhibit fast activation-deactivation switching. Among optogenetic tools, long-wavelength light-controlled systems are more preferable for use in mammalian tissues in comparison with tools utilizing shorter wavelengths, since far-red/near-infrared light has the maximum penetration depth due to lower light scattering caused by lipids and reduced tissue autofluorescence at wavelengths above 700 nm. Here, we review such light-inducible systems, which are based on synthetic factors that can be targeted to any desired DNA sequence and provide activation or repression of a gene of interest. The factors include zinc finger proteins, transcription activator-like effectors (TALEs), and the CRISPR/Cas9 technology. We also discuss the advantages and disadvantages of these DNA targeting tools in the context of the light-inducible gene regulation systems.

scholarly journals Screening and evaluation of the strong endogenous promoters in Pichia pastoris

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Weiwang Dou ◽  
Quanchao Zhu ◽  
Meihua Zhang ◽  
Zuyuan Jia ◽  
Wenjun Guan
Keyword(s):  
Pichia Pastoris ◽  
Protein Expression ◽  
Stationary Phase ◽  
Expression Level ◽  
Logarithmic Phase ◽  
Competitive Effect ◽  
Transcriptional Expression ◽  
Promoter Elements ◽  
Endogenous Promoter ◽  
Β Carotene

Abstract Background Due to its ability to perform fast and high-density fermentation, Pichia pastoris is not only used as an excellent host for heterologous protein expression but also exhibits good potential for efficient biosynthesis of small-molecule compounds. However, basic research on P. pastoris lags far behind Saccharomyces cerevisiae, resulting in a lack of available biological elements. Especially, fewer strong endogenous promoter elements available for foreign protein expression or construction of biosynthetic pathways were carefully evaluated in P. pastoris. Thus, it will be necessary to identify more available endogenous promoters from P. pastoris. Results Based on RNA-seq and LacZ reporter system, eight strong endogenous promoters contributing to higher transcriptional expression levels and β-galactosidase activities in three frequently-used media were screened out. Among them, the transcriptional expression level contributed by P0019, P0107, P0230, P0392, or P0785 was basically unchanged during the logarithmic phase and stationary phase of growth. And the transcriptional level contributed by P0208 or P0627 exhibited a growth-dependent characteristic (a lower expression level during the logarithmic phase and a higher expression level during the stationary phase). After 60 h growth, the β-galactosidase activity contributed by P0208, P0627, P0019, P0407, P0392, P0230, P0785, or P0107 was relatively lower than PGAP but higher than PACT1. To evaluate the availability of these promoters, several of them were randomly applied to a heterogenous β-carotene biosynthetic pathway in P. pastoris, and the highest yield of β-carotene from these mutants was up to 1.07 mg/g. In addition, simultaneously using the same promoter multiple times could result in a notable competitive effect, which might significantly lower the transcriptional expression level of the target gene. Conclusions The novel strong endogenous promoter identified in this study adds to the number of promoter elements available in P. pastoris. And the competitive effect observed here suggests that a careful pre-evaluation is needed when simultaneously and multiply using the same promoter in one yeast strain. This work also provides an effective strategy to identify more novel biological elements for engineering applications in P. pastoris.

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