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Applied Microbiology and Biotechnology Feb 2024Metabolic engineering frequently makes use of point mutation and saturation mutation library creation. At present, sequencing is the only reliable and direct technique...
Metabolic engineering frequently makes use of point mutation and saturation mutation library creation. At present, sequencing is the only reliable and direct technique to detect point mutation and screen saturation mutation library. In this study, mismatch amplification mutation assay (MAMA) PCR was used to detect point mutation and screen saturation mutation library. In order to fine-tune the expression of odhA encoding 2-oxoglutarate dehydrogenase E1 component, a saturating mutant library of the RBS of odhA was created in Corynebacterium glutamicum P12 based on the CRISPR-Cas2a genome editing system, which increased the L-proline production by 81.3%. MAMA PCR was used to filter out 42% of the non-mutant transformants in the mutant library, which effectively reduced the workload of the subsequent fermentation test and the number of sequenced samples. The rapid and sensitive MAMA-PCR method established in this study provides a general strategy for detecting point mutations and improving the efficiency of mutation library screening. KEY POINTS: • MAMA PCR was optimized and developed to detect point mutation. • MAMA PCR greatly improves the screening efficiency of point mutation. • Attenuation of odhA expression in P12 effectively improves proline production.
Topics: Point Mutation; Mutation; Base Sequence; Corynebacterium glutamicum; Polymerase Chain Reaction
PubMed: 38305911
DOI: 10.1007/s00253-024-13036-2 -
Frontiers in Immunology 2022Sphingosine-1-phosphate lyase is encoded by the gene, degrades S1P, and is crucial for S1P homeostasis in animal models and humans. S1P lyase deficient patients suffer...
Sphingosine-1-phosphate lyase is encoded by the gene, degrades S1P, and is crucial for S1P homeostasis in animal models and humans. S1P lyase deficient patients suffer from adrenal insufficiency, severe lymphopenia, and skin disorders. In this study, we used random mutagenesis screening to identify a mouse line carrying a missense mutation of (M467K). This mutation caused similar pathologies as Sgpl1 knock-out mice in multiple organs, but greatly preserved its lifespan, which M467K mutation mice look normal under SPF conditions for over 40 weeks, in contrast, the knock-out mice live no more than 6 weeks. When treated with Imiquimod, mice experienced exacerbated skin inflammation, as revealed by aggravated acanthosis and orthokeratotic hyperkeratosis. We also demonstrated that the IL17a producing Vγ6 cell was enriched in skin and caused severe pathology after imiquimod treatment. Interestingly, hyperchromic plaque occurred in the mutant mice one month after Imiquimod treatment but not in the controls, which resembled the skin disorder found in deficient patients. Therefore, our results demonstrate that point mutation mice successfully modeled a human disease after being treated with Imiquimod. We also revealed a major subset of γδT cells in the skin, IL17 secreting Vγ6 T cells were augmented by deficiency and led to skin pathology. Therefore, we have, for the first time, linked the IL17a and γδT cells to SPL insufficiency.
Topics: Animals; Homeostasis; Hyperpigmentation; Imiquimod; Mice; Mice, Knockout; Point Mutation
PubMed: 35769463
DOI: 10.3389/fimmu.2022.728455 -
European Journal of Immunology Oct 2022IRF4 knock-out cells do not differentiate properly into T helper subsets 2, 9, and 17. Compared to WT IRF4 point mutation L116R shows differentially expressed key...
IRF4 knock-out cells do not differentiate properly into T helper subsets 2, 9, and 17. Compared to WT IRF4 point mutation L116R shows differentially expressed key cytokines. While amounts Th2 and Th17 cells are decreased, there is a strong induction of Th9 cells in cells carrying the L116R mutated IRF4.
Topics: Cell Differentiation; Cytokines; Interferon Regulatory Factors; Interferons; Interleukin-9; Point Mutation; Th17 Cells; Th2 Cells
PubMed: 36047738
DOI: 10.1002/eji.202249869 -
Molecular Metabolism Dec 2021Protein disulfide isomerases (PDIs) are oxidoreductases that are involved in catalyzing the formation and rearrangement of disulfide bonds during protein folding. One of...
OBJECTIVE
Protein disulfide isomerases (PDIs) are oxidoreductases that are involved in catalyzing the formation and rearrangement of disulfide bonds during protein folding. One of the PDI members is the PDI-associated 6 (PDIA6) protein, which has been shown to play a vital role in β-cell dysfunction and diabetes. However, very little is known about the function of this protein in β-cells in vivo. This study aimed to describe the consequences of a point mutation in Pdia6 on β-cell development and function.
METHODS
We generated an ENU mouse model carrying a missense mutation (Phe175Ser) in the second thioredoxin domain of the Pdia6 gene. Using biochemical and molecular tools, we determined the effects of the mutation on the β-cell development at embryonic day (E)18.5 and β-cell identity as well as function at postnatal stages.
RESULTS
Mice homozygous for the Phe175Ser (F175S) mutation were mildly hyperglycemic at weaning and subsequently became hypoinsulinemic and overtly diabetic at the adult stage. Although no developmental phenotype was detected during embryogenesis, mutant mice displayed reduced insulin-expressing β-cells at P14 and P21 without any changes in the rate of cell death and proliferation. Further analysis revealed an increase in BiP and the PDI family member PDIA4, but without any concomitant apoptosis and cell death. Instead, the expression of prominent markers of β-cell maturation and function, such as Ins2, Mafa, and Slc2a2, along with increased expression of α-cell markers, Mafb, and glucagon was observed in adult mice, suggesting loss of β-cell identity.
CONCLUSIONS
The results demonstrate that a global Pdia6 mutation renders mice hypoinsulinemic and hyperglycemic. This occurs due to the loss of pancreatic β-cell function and identity, suggesting a critical role of PDIA6 specifically for β-cells.
Topics: Animals; Diabetes Mellitus; Insulin-Secreting Cells; Mice; Mice, Inbred C3H; Point Mutation; Protein Disulfide-Isomerases
PubMed: 34487921
DOI: 10.1016/j.molmet.2021.101334 -
ACS Synthetic Biology Jun 2021Minicells are nanosized membrane vesicles produced by bacteria. Minicells are chromosome-free but contain cellular biosynthetic and metabolic machinery, and they are...
Minicells are nanosized membrane vesicles produced by bacteria. Minicells are chromosome-free but contain cellular biosynthetic and metabolic machinery, and they are robust due to the protection provided by the bacterial cell envelope, which makes them potentially highly attractive in biomedical applications. However, the applicability of minicells and other nanoparticle-based delivery systems is limited by their inefficient accumulation at the target. Here we engineered the minicell-producing strain to overexpress flagellar genes, which enables the generation of motile minicells. We subsequently performed an experimental and theoretical analysis of the minicell motility and their responses to gradients of chemoeffectors. Despite important differences between the motility of minicells and normal bacterial cells, minicells were able to bias their movement in chemical gradients and to accumulate toward the sources of chemoattractants. Such motile and chemotactic minicells may thus be applicable for an active effector delivery and specific targeting of tissues and cells according to their metabolic profiles.
Topics: Chemotactic Factors; Chemotaxis; Cytoplasmic Vesicles; Drug Delivery Systems; Escherichia coli; Flagella; Gene Expression; Genetic Engineering; Microorganisms, Genetically-Modified; Movement; Nanoparticles; Point Mutation
PubMed: 34081866
DOI: 10.1021/acssynbio.1c00012 -
BMC Biology Oct 2023Gene knockout and knock-in have been widely performed in large farm animals based on genome editing systems. However, many types of precise gene editing, including...
BACKGROUND
Gene knockout and knock-in have been widely performed in large farm animals based on genome editing systems. However, many types of precise gene editing, including targeted deletion, gene tagging, and large gene fragment replacement, remain a challenge in large farm animals.
RESULTS
Here, we established versatile self-excising gene-targeting technology in combination with programmable nucleases (SEGCPN) to efficiently generate various types of precise gene editing in bovine. First, we used this versatile method to successfully generate bovine embryos with point mutations and 11-bp deletions at the MSTN locus. Second, we successfully generated bulls with EGFP labeling at the SRY locus. Finally, we successfully generated humanized cows in which the endogenous 18-kb α-casein gene was replaced with a 2.6-kb human α-lactalbumin gene.
CONCLUSIONS
In summary, our new SEGCPN method offers unlimited possibilities for various types of precise gene editing in large animals for application both in agriculture and disease models.
Topics: Female; Animals; Cattle; Male; Humans; Gene Editing; CRISPR-Cas Systems; Gene Targeting; Gene Knockout Techniques; Point Mutation
PubMed: 37864194
DOI: 10.1186/s12915-023-01677-0 -
Genes Apr 2023Cytosine base editors (CBEs) and adenine base editors (ABEs) are recently developed CRISPR-mediated genome-editing tools that do not introduce double-strand breaks. In...
Cytosine base editors (CBEs) and adenine base editors (ABEs) are recently developed CRISPR-mediated genome-editing tools that do not introduce double-strand breaks. In this study, five ABEs, ABE7.10, ABEmax, NG-ABEmax, ABE8e and NG-ABE8e, were used to generate A-to-G (T-to-C) conversions in five genome loci in porcine fetal fibroblasts (PFFs). Variable yet appreciable editing efficiencies and variable activity windows were observed in these targeting regions via these five editors. The strategy of two sgRNAs in one vector exhibited superior editing efficiency to that of using two separate sgRNA expression vectors. ABE-mediated start-codon mutation in silenced its expression of protein and, unexpectedly, eliminated the vast majority of its mRNA. No off-target DNA site was detected for these editors. Substantial off-target RNA events were present in the ABE-edited cells, but no KEGG pathway was found to be significantly enriched. Our study supports that ABEs are powerful tools for A-to-G (T-to-C) point-mutation modification in porcine cells.
Topics: Animals; Swine; Adenine; Gene Editing; Mutation; Point Mutation; Fibroblasts
PubMed: 37107666
DOI: 10.3390/genes14040908 -
Archives of Razi Institute Mar 2020Growth differentiation factor 9 (GDF9) plays a critical role in ovarian follicular development and ovulation rate. The present study aimed to investigate the correlation...
Growth differentiation factor 9 (GDF9) plays a critical role in ovarian follicular development and ovulation rate. The present study aimed to investigate the correlation between the single-nucleotide polymorphism (SNP) of the GDF9 gene and reproductive performance variables, such as fertility and sterility in Awassi sheep. Forty pairs of ovaries from a total of 40 slaughtered Iraqi Awassi ewes were used in this study. Twenty of the ovaries were collected from sterile ewes and the other 20 ovaries were taken from fertile ewes for genomic DNA extraction, polymerase chain reaction, and sequencing to detect GDF9 gene polymorphism. Follicles and oocytes of all the 40 ovaries were evaluated and compared with the results of genotyping. Furthermore, histopathological and microscopic evaluations were performed for 40 ovarian tissues of the two groups. The sequence analysis revealed that exon I had three SNPs, including T(114)C, G(129)R, and G(199)A. The first two SNPs were silent mutations and the last mutation was missense responsible for the substitution of glutamic acid with lysine at position 67. The current study showed a significant increase (P≤0.01) in GG, AA, CC, GA, and GG genotypes at G(129)R, G(199)A, T(114)C, G(129)R, and G(199)A loci, respectively. Moreover, the TT genotype in locus T(114)C was recorded to significantly augment (P≤0.05) in the fertile ewes. Mutant GA genotype of the G(129)R locus led to a significant (P≤0.05) increase in the percentage of follicles (4-8 mm) and oocytes number, compared to wild GG. On the other hand, a significant decrease was recorded in the mutant AA genotype in G(199)A, compared to wild GG. Differences between CC and TT genotypes at T(114)C locus were not significant. Histopathological examination revealed hypoplasia in the ovarian tissue of sterile ewes accompanied by fibrous connective tissue invasion and follicles degeneration. However, in the fertile ewes, the ovarian tissues were normal with the presence of numerous corpus albicans and degenerative corpus luteum. According to the findings of this study, the homozygote mutation in fertile ewes minimized the number of follicles and oocytes leading to sterility, while the heterozygote mutation was reported in the fertile Awassi ewes.
Topics: Animals; Female; Fertility; Growth Differentiation Factor 9; Infertility; Oocytes; Point Mutation; Sheep; Sheep Diseases; Sheep, Domestic
PubMed: 32292008
DOI: 10.22092/ari.2018.122232.1220 -
PloS One 2020Adenocarcinoma is the most common type of non-small cell lung cancer. Some causative genomic alterations in epidermal growth factor receptor (EGFR), including deletions...
Adenocarcinoma is the most common type of non-small cell lung cancer. Some causative genomic alterations in epidermal growth factor receptor (EGFR), including deletions in exon 19 (E19 dels) and a point mutation in E21, are known to have favourable prognoses due to sensitivity to tyrosine kinase inhibitors; however, the prognoses of other uncommon mutations are unclear. This study analysed the clinical significance of EGFR mutation types in lung adenocarcinoma. We retrospectively reviewed 1,020 subjects (mean age: 66.8 years, female: 41.7%) who were diagnosed with advanced lung adenocarcinoma, had EGFR mutation data, and did not undergo surgery from five medical institutes between 2010 and 2016. Subjects were classified according to EGFR mutation status, particularly for exon-specific mutations. EGFR positivity was defined as the presence of mutation and EGFR negativity was defined as wild-type EGFR. EGFR positivity was 38.0%, with the incidence of mutations in E18, E19, E20, and E21 was 3.6%, 51.0%, 3.4%, and 42.0%, respectively. The EGFR positive group survived significantly longer than the negative group (p<0.001), and there was a significant difference in survival among the four EGFR mutation sites (p = 0.003); E19 dels were the only significant factor that lowered mortality (HR: 0.678, p = 0.002), while an E21 mutation was the prognostic factor associated with the most increased mortality (HR: 1.365, p = 0.015). Amongst EGFR positive subjects, the proportion of E19 dels in TKI-responders was significantly higher and that of E21 mutations significantly lower, compared with non-responders. In TKI treatment, mutations in E18 and E20 were not worse factors than the E21 L858R mutation. In conclusion, the presence of EGFR mutations in advanced lung adenocarcinoma can predict a good prognosis; E19 dels prospect to have a better prognosis than other mutations, while an E21 mutation is expected to increase mortality.
Topics: Adenocarcinoma of Lung; Aged; Asian People; Carcinoma, Non-Small-Cell Lung; ErbB Receptors; Exons; Female; Humans; Lung Neoplasms; Male; Middle Aged; Mutation; Point Mutation; Prognosis; Republic of Korea; Retrospective Studies; Sequence Deletion
PubMed: 32053675
DOI: 10.1371/journal.pone.0228925 -
Brain : a Journal of Neurology Oct 2022This scientific commentary refers to ‘The impact of phosphorylated PTEN at threonine 366 on cortical connectivity and behaviour’ by Ledderose...
This scientific commentary refers to ‘The impact of phosphorylated PTEN at threonine 366 on cortical connectivity and behaviour’ by Ledderose (https://doi.org/10.1093/brain/awac188).
Topics: Humans; Phosphorylation; Point Mutation; Threonine; PTEN Phosphohydrolase
PubMed: 36148582
DOI: 10.1093/brain/awac350