CRISPR/Cas9-induced β-carotene hydroxylase mutation in Dunaliella salina CCAP19/18

Abstract Dunaliella salina (D. salina) has been exploited as a novel expression system for the field of genetic engineering. However, owing to the low or inconsistent expression of target proteins, it has been greatly restricted to practical production of recombinant proteins. Since the accurate gene editing function of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system, β-carotene hydroxylase gene was chosen as an example to explore D. salina application with the purpose of improving expression level of foreign genes. In this paper, based on pKSE401 backbone, three CRISPR/Cas9 binary vectors were constructed to targeting exon 1 and 3 of the β-carotene hydroxylase of D. salina CCAP19/18 (Dschyb). D. salina mutants were obtained by salt gradient transformation method, and the expression of Dschyb gene were identified through real-time fluorescent quantitative PCR. Moreover, carotenoids content was analyzed by high-performance liquid chromatography at different time points after high intensity treatment. Compared with wild type strains, the β-carotene levels of mutants showed a significant increase, nearly up to 1.4 μg/ml, and the levels of zeaxanthin decreased to various degrees in mutants. All the results provide a compelling evidence for targeted gene editing in D. salina. This study gave a first successful gene editing of D. salina which has a very important practical significance for increasing carotene yield and meeting realistic industry demand. Furthermore, it provides an approach to overcome the current obstacles of D. salina, and then gives a strong tool to facilitates the development and application of D. salina system

Cryptosporidiosis in broiler chickens in Zhejiang Province, China: molecular characterization of oocysts detected in fecal samples

Cryptosporidium is one of the most important parasites in poultry, and this pathogen can infect more than 30 avian species. The present study investigated the infection rate of Cryptosporidium among broiler chicken flocks. A total of 385 fecal samples from broiler chickens in 7 regions of Zhejiang Province collected from November 2010 to January 2012 were examined by microscopy. Thirty-eight (10%) samples were positive for Cryptosporidium infection, and 3 genotypes (Cryptosporidium baileyi, Cryptosporidium meleagridis, and avian genotype II) were identified by PCR and sequencing. A phylogenetic tree of the isolates was analyzed. These results suggest that cryptosporidiosis is widespread in poultry in Zhejiang Province, and is a potential threat to public health as well as the economy. This is the first report about the infection rate and molecular characterization of Cryptosporidium in broiler chickens in Zhejiang

Characterization of the development of Haemonchus contortus ZJ strain from gerbils

Abstract Background Haemonchus contortus is a serious parasitic nematode in domestic ruminants around the world, including China. Haemonchus contortus has developed extensive resistance to commercial anthelmintics, which has produced a demand for new control methods, such as more effective drugs. Gerbils infected with H. contortus have previously been used as a model for anthelmintics selections, and the growth of H. contortus had been briefly examined. To enhance the model, this study provides an additional description of the development of H. contortus ZJ strain in gerbils. Results Gerbils were infected with H. contortus ZJ strain at a dose of 2000 exsheathed infective larvae (xL3s) and sacrificed at 4, 7 and 18 days post-infection (dpi). Only fourth-stage larvae were found in the stomachs. About 2% of the inoculums were obtained at each of the three sampling time points. Larvae grew more slowly in gerbils than in sheep, but presented almost the same morphology. Rod-like crystalline inclusions were present in the intestinal cells of larvae, indicating that the metabolic rate of larvae was probably greatly reduced. Histological examination of stomach sections showed that larvae are located in the lumens or at the mucosal surfaces, with few inflammatory changes evident. Conclusions The development and features of H. contortus ZJ strain in gerbils were described. Our results provide supplementary information of H. contortus growth in gerbils, especially the presence of rod-like crystalline inclusions, and may contribute to improve the anthelmintic selection system

Characterization of Anti-Citrinin Specific ScFvs Selected from Non-Immunized Mouse Splenocytes by Eukaryotic Ribosome Display.

Single chain variable fragments (scFvs) against citrinin (CIT) were selected from a scFv library constructed from the splenocytes of non-immunized mice by an improved eukaryotic ribosome display technology in this study. Bovine serum albumin (BSA)/ CIT-BSA and ovalbumin (OVA)/ CIT-OVA were used as the antigens to select specific anti-CIT scFvs. Eukaryotic in situ RT-PCR method was used to recover the selected mRNA after every affinity selection. After six rounds of ribosome display, expression vector pTIG-TRX carrying specific scFv DNAs were constructed and transformed into Escherichia coli BL21 (DE3) for protein expression. Thirteen positive clones were selected out of which three (designated 23, 68 and 109) showed high binding activity and specificity to CIT by indirect ELISA, while no clone showed binding activity with carrier proteins. The three scFvs showed high specificity to CIT and the cross reactivity with other mycotoxins was below 0.01% as determined by indirect competitive ELISA. These specific scFvs offer a potential novel immunoassay method for CIT residues. This study confirmed the effectiveness of the improved eukaryotic ribosome display system and could be used as a reference for the selection of scFvs specific to other small molecules using ribosome display

The heat shock protein 90 of Toxoplasma gondii is essential for invasion of host cells and tachyzoite growth

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects almost all warm-blooded vertebrates. Heat shock proteins (HSP) regulate key signal transduction events in many organisms, and heat shock protein 90 (Hsp90) plays an important role in growth, development, and virulence in several parasitic protozoa. Here, we discovered increased transcription of the Hsp90 gene under conditions for bradyzoite differentiation, i.e. alkaline and heat shock conditions in vitro, suggesting that Hsp90 may be connected with bradyzoite development in T. gondii. A knockout of the TgHsp90 strain (ΔHsp90) and a complementation strain were constructed. The TgHsp90 knockout cells were found to be defective in host-cell invasion, were not able to proliferate in vitro in Vero cells, and did not show long-time survival in mice in vivo. These inabilities of the knockout parasites were restored upon complementation of TgHsp90. These data unequivocally show that TgHsp90 contributes to bradyzoite development, and to invasion and replication of T. gondii in host cells

The heat shock protein 90 of

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects almost all warm-blooded vertebrates. Heat shock proteins (HSP) regulate key signal transduction events in many organisms, and heat shock protein 90 (Hsp90) plays an important role in growth, development, and virulence in several parasitic protozoa. Here, we discovered increased transcription of the Hsp90 gene under conditions for bradyzoite differentiation, i.e. alkaline and heat shock conditions in vitro, suggesting that Hsp90 may be connected with bradyzoite development in T. gondii. A knockout of the TgHsp90 strain (ΔHsp90) and a complementation strain were constructed. The TgHsp90 knockout cells were found to be defective in host-cell invasion, were not able to proliferate in vitro in Vero cells, and did not show long-time survival in mice in vivo. These inabilities of the knockout parasites were restored upon complementation of TgHsp90. These data unequivocally show that TgHsp90 contributes to bradyzoite development, and to invasion and replication of T. gondii in host cells

Phosphorylation of Threonine343 Is Crucial for OCT4 Interaction with SOX2 in the Maintenance of Mouse Embryonic Stem Cell Pluripotency

Summary: OCT4 is required to maintain the pluripotency of embryonic stem cells (ESCs); yet, overdose-expression of OCT4 induces ESC differentiation toward primitive endoderm. The molecular mechanism underlying this differentiation switch is not fully understood. Here, we found that substitution of threonine343 by alanine (T343A), but not aspartic acid (T343D), caused a significant loss of OCT4-phosphorylation signal in ESCs. Loss of such OCT4-phosphorylation compromises its interaction with SOX2 but promotes interaction with SOX17. We therefore propose that threonine343-based OCT4-phosphorylation is crucial for the maintenance of ESC pluripotency. This OCT4-phosphorylation-based mechanism may provide insight into the regulation of lineage specification during early embryonic development. : Li and colleagues show that phosphorylation of threonine343 in OCT4 mediates global OCT4-phosphorylation (phos-OCT4T343). Phos-OCT4T343 is crucial for OCT4 to protect embryonic stem cell (ESC) pluripotency. Furthermore, phos-OCT4T343 interacts with SOX2 but non-phos-OCT4T343 interacts with SOX17 in the regulation of ESC pluripotency and differentiation. It also suggests a mechanism for lineage commitment during early embryonic development. Keywords: OCT4-phosphorylation, ESC pluripotency, lineage specification, genome editing, protein interaction, SOX2, SOX17, embryonic developmen

Site-Directed Mutagenesis Study Revealed Three Important Residues in Hc-DAF-22, a Key Enzyme Regulating Diapause of Haemonchus contortus

Haemonchus contortus (H. contortus) is one of the most important parasites of small ruminants, especially goats and sheep. The complex life cycle of this nematode is a main obstacle for the control and prevention of haemonchosis. So far, a special form of arrested development called diapause different from the dauer stage in Caenorhabditis elegans (C. elegans) has been found in many parasitic nematodes. In our previous study, we have characterized a novel gene Hc-daf-22 from H. contortus sharing high homology with Ce-daf-22 and functional analysis showed this gene has similar biological function with Ce-daf-22. In this study, Hc-daf-22 mutants were constructed using site-directed mutagenesis, and carried out rescue experiments, RNA interference (RNAi) experiments and in vitro enzyme activity analysis with the mutants to further explore the precise function site of Hc-DAF-22. The results showed that Hc-daf-22 mutants could be expressed in the rescued ok693 worms and the expression positions were mainly in the intestine which was identical with that of Hc-daf-22 rescued worms. Through lipid staining we found that Hc-daf-22 could rescue daf-22 mutant (ok693) from the fatty acid metabolism deficiency while Hc-daf-22 mutants failed. Brood size and body length analyses in rescue experiment along with body length and life span analyses in RNAi experiment elucidated that Hc-daf-22 resembled Ce-daf-22 in effecting the development and capacity of C. elegans and mutants impaired the function of Hc-daf-22. Together with the protease activity assay, this research revealed three important active resides 84C/299H/349H in Hc-DAF-22 by site-directed mutagenesis