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A Potential Method for Rapid Screening of Amphioxus Founder Harboring Germline Mutation and Transgene.

ABSTRACT: Amphioxus is a promising model organism for understanding the origin and evolution of vertebrates due to its basal phylogenetic position among chordates. We here compared the mutation efficacy and mutation type of tail tips and gametes of amphioxus founders injected with Cas9 protein and six different sgRNAs targeting five distinct genes, and revealed a strong correlation for mutation efficacy and a mild correlation for mutation type among the two tissues. In addition, we also observed a positive relationship between gene insertions observed in tail tips and gametes of amphioxus founders injected with Tol2 transposase and two different transgenic constructs. Finally, we showed that amphioxus larvae which had their tail tips cut at the 3-4 gill-slit stage were able to recover within 6 days and developed a normal number of gonads at the adult stage, and that F0 larvae carry similar mutation efficacy and type in the posterior end to that in the tail tips after their metamorphosis. Together, these findings suggest a great potential for obtaining valid amphioxus founders with desired mutations and transgenes at as early as the early larval stage, which will certainly speed up the generation of amphioxus mutants and transgenes and make it more cost- and labor-effective.

SUBMITTER: Zou J

PROVIDER: S-EPMC8387717 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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A Potential Method for Rapid Screening of Amphioxus Founder Harboring Germline Mutation and Transgene.

Zou Jiaqi J Wu Xiaotong X Shi Chenggang C Zhong Yanhong Y Zhang Lei L Yan Qiuning Q Su Liuru L Li Guang G

Frontiers in cell and developmental biology 20210812

Amphioxus is a promising model organism for understanding the origin and evolution of vertebrates due to its basal phylogenetic position among chordates. We here compared the mutation efficacy and mutation type of tail tips and gametes of amphioxus founders injected with Cas9 protein and six different sgRNAs targeting five distinct genes, and revealed a strong correlation for mutation efficacy and a mild correlation for mutation type among the two tissues. In addition, we also observed a positiv ...[more]

PMID: 34458263

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Project description:This study aimed to investigate the PCR-based screening strategy for the prediction of the antimicrobial biosynthetic potential of the selected Streptomyces strains originated from an extreme environment (Cholistan Desert, Pakistan). The biosynthetic potential was determined by using both molecular and culture-dependent screening approaches. The four biosynthetic genes clusters, including the pks-1, nrps, cyp P450 hydroxylase (cyps), and glycopeptide oxy b genes, were investigated in the selected strains by PCR amplification, sequencing, and by subsequent bioinformatics approaches. Among the 40 selected Streptomyces strains, 33 strains possessed the nrps gene, 17 strains carried the pks-1 gene, four strains were found to have the cyps gene, and none of the strain carried oxy b gene. The Streptomyces strains including NR-1, NR-10, NR-14, and NR-15 were investigated for in vitro antifungal activity against Fusarium oxysporum, Rhizoctonia solani, and Aspergillus sp. The extracts were analyzed for chemical profiling (TLC and HPLC-UV), and a unique pattern of secondary metabolites was observed. The selected strains exhibited pronounced antifungal activity against the fungal test strains with the zone of inhibition up to 17, 18, and 19 mm, respectively. The study depicts that gene-based screening can be successfully applied to identify potentially bioactive strains by usin a single screening process. This PCR-based approach is rapid and can be used for sorting out and selecting the potential candidate among actinobacterial culture collections. Such a preselection or strain prioritization consequently decreases the time and efforts required for selecting the potential bioactive strain, which then can be subjected to the detailed chemical analysis.This study aimed to investigate the PCR-based screening strategy for the prediction of the antimicrobial biosynthetic potential of the selected Streptomyces strains originated from an extreme environment (Cholistan Desert, Pakistan). The biosynthetic potential was determined by using both molecular and culture-dependent screening approaches. The four biosynthetic genes clusters, including the pks-1, nrps, cyp P450 hydroxylase (cyps), and glycopeptide oxy b genes, were investigated in the selected strains by PCR amplification, sequencing, and by subsequent bioinformatics approaches. Among the 40 selected Streptomyces strains, 33 strains possessed the nrps gene, 17 strains carried the pks-1 gene, four strains were found to have the cyps gene, and none of the strain carried oxy b gene. The Streptomyces strains including NR-1, NR-10, NR-14, and NR-15 were investigated for in vitro antifungal activity against Fusarium oxysporum, Rhizoctonia solani, and Aspergillus sp. The extracts were analyzed for chemical profiling (TLC and HPLC-UV), and a unique pattern of secondary metabolites was observed. The selected strains exhibited pronounced antifungal activity against the fungal test strains with the zone of inhibition up to 17, 18, and 19 mm, respectively. The study depicts that gene-based screening can be successfully applied to identify potentially bioactive strains by usin a single screening process. This PCR-based approach is rapid and can be used for sorting out and selecting the potential candidate among actinobacterial culture collections. Such a preselection or strain prioritization consequently decreases the time and efforts required for selecting the potential bioactive strain, which then can be subjected to the detailed chemical analysis.

Dataset Information

A Potential Method for Rapid Screening of Amphioxus Founder Harboring Germline Mutation and Transgene.

Publications

A Potential Method for Rapid Screening of Amphioxus Founder Harboring Germline Mutation and Transgene.

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