Interactome Mapping of eIF3A in a Colon Cancer and an Immortalized Embryonic Cell Line Using Proximity-Dependent Biotin Identification

Translation initiation comprises complex interactions of eukaryotic initiation factor (eIF) subunits and the structural elements of the mRNAs. Translation initiation is a key process for building the cell’s proteome. It not only determines the total amount of protein synthesized but also controls the translation efficiency for individual transcripts, which is important for cancer or ageing. Thus, understanding protein interactions during translation initiation is one key that contributes to understanding how the eIF subunit composition influences translation or other pathways not yet attributed to eIFs. We applied the BioID technique to two rapidly dividing cell lines (the immortalized embryonic cell line HEK-293T and the colon carcinoma cell line HCT-166) in order to identify interacting proteins of eIF3A, a core subunit of the eukaryotic initiation factor 3 complex. We identified a total of 84 interacting proteins, with very few proteins being specific to one cell line. When protein biosynthesis was blocked by thapsigargin-induced endoplasmic reticulum (ER) stress, the interacting proteins were considerably smaller in number. In terms of gene ontology, although eIF3A interactors are mainly part of the translation machinery, protein folding and RNA binding were also found. Cells suffering from ER-stress show a few remaining interactors which are mainly ribosomal proteins or involved in RNA-binding.

Derivation and Characterization of a New Embryonic Cell Line from the Olive Flounder Paralichthys olivaceus

A new embryonic cell line that consisted predominantly of fibroblast cells has been established from embryos at Kupffer’s vesicle (KV) stage of the olive flounder Paralichthys olivaceus, designated as the PoEKC. PoEKC cells have been subcultured for 61 passages over a period of 18 months. The cells were cultured in DMEM/F-12 medium supplemented with antibiotics, FBS, and growth factors at temperature of 25°C. The growth curve of the PoEKC cells comprised an exponential growth phase and a long plateau phase. Chromosome analysis revealed that the cells possessed the normal flounder diploid karyotype of 2n = 48t. The origin of the cell line was confirmed by testing the partial sequences of cytochrome oxidase c subunit I (COI) gene of mtDNA. The pluripotency markers, genes OCT4-1, SOX2, KLF4, and NANOG showed positive signals in the PoEKC cells, and the cells also presented high ALP activity. According to the above results, the PoEKC cells might be pluripotency, though the pluripotency needs further confirmation. The cells were also successfully transfected with GFP reporter gene suggesting that it could be utilized for gene function study in the flounder.

Diminished adherence of Biomphalaria glabrata embryonic cell line to sporocysts of Schistosoma mansoni following programmed knockout of the allograft inflammatory factor

Background Larval development in an intermediate host gastropod snail of the genus Biomphalaria is an obligatory component of the life-cycle of Schistosoma mansoni. Understanding of the mechanism(s) of host defense may hasten the development of tools that block transmission of schistosomiasis. The allograft inflammatory factor 1, AIF, which is evolutionarily conserved and expressed in phagocytes, is a marker of macrophage activation in both mammals and invertebrates. AIF enhances cell proliferation and migration. The embryonic cell line, termed Bge, from Biomphalaria glabrata is a versatile resource for investigation of the snail-schistosome relationship since Bge exhibits a hemocyte-like phenotype. Hemocytes perform central roles in innate and cellular immunity in gastropods and in some cases can kill the parasite. However, the Bge cells do not kill the parasite in vitro. Methods Bge cells were transfected by electroporation with plasmid pCas-BgAIFx4, encoding the Cas9 nuclease and a guide RNA specific for exon 4 of the B. glabrata AIF (BgAIF) gene. Transcript levels for Cas9 and for BgAIF were monitored by reverse-transcription-PCR and, in parallel, adhesion of gene-edited Bge cells during co-culture with of schistosome sporocysts was assessed. Results Gene knockout manipulation induced gene-disrupting indels, frequently 1–2 bp insertions and/or 8–30 bp deletions, at the programmed target site; a range from 9 to 17% of the copies of the BgAIF gene in the Bge population of cells were mutated. Transcript levels for BgAIF were reduced by up to 73% (49.5 ± 20.2% SD, P ≤ 0.05, n = 12). Adherence by BgAIF gene-edited (ΔBgAIF) Bge to sporocysts diminished in comparison to wild type cells, although cell morphology did not change. Specifically, as scored by a semi-quantitative cell adherence index (CAI), fewer ΔBgAIF than control wild type cells adhered to sporocysts; control CAI, 2.66 ± 0.10, ΔBgAIF, 2.30 ± 0.22 (P ≤ 0.01). Conclusions The findings supported the hypothesis that BgAIF plays a role in the adherence of B. glabrata hemocytes to sporocysts during schistosome infection in vitro. This demonstration of the activity of programmed gene editing will enable functional genomics approaches using CRISPR/Cas9 to investigate additional components of the snail-schistosome host-parasite relationship.

Diminished adherence of Biomphalaria glabrata embryonic cell line to sporocysts of Schistosoma mansoni following programmed knockout of the allograft inflammatory factor

Background: Larval development in an intermediate host gastropod snail of the genus Biomphalaria is an obligatory component of the life-cycle of Schistosoma mansoni. Understanding of the mechanism(s) of host defense may hasten the development of tools that block transmission of schistosomiasis. The allograft inflammatory factor 1, AIF, which is evolutionarily conserved and expressed in phagocytes, is a marker of macrophage activation in both mammals and invertebrates. AIF enhances cell proliferation and migration. The embryonic cell line, termed Bge, from Biomphalaria glabrata is a versatile resource for investigation of the snail-schistosome relationship since Bge exhibits a hemocyte-like phenotype. Hemocytes perform central roles in innate and cellular immunity in gastropods and in some cases can kill the parasite. However, the Bge cells do not kill the parasite in vitro.Methods: Bge cells were transfected by electroporation with plasmid pCas-BgAIFx4, encoding the Cas9 nuclease and a guide RNA specific for exon 4 of the B. glabrata AIF (BgAIF) gene. Transcript levels for Cas9 and for BgAIF were monitored by reverse-transcription-PCR and, in parallel, adhesion of gene-edited Bge cells during co-culture with of schistosome sporocysts was assessed.Results: Gene knockout manipulation induced gene-disrupting indels, frequently 1–2 bp insertions and/or 8–30 bp deletions, at the programmed target site; a range from 9 to 17% of the copies of the BgAIF gene in the Bge population of cells were mutated. Transcript levels for BgAIF were reduced by up to 73% (49.5 ± 20.2% SD, P ≤ 0.05, n =12). Adherence by BgAIF gene-edited (ΔBgAIF) Bge to sporocysts diminished in comparison to wild type cells, although cell morphology did not change. Specifically, as scored by a semi-quantitative cell adherence index (CAI), fewer ΔBgAIF than control wild type cells adhered to sporocysts; control CAI, 2.66 ± 0.10, ΔBgAIF, 2.30 ± 0.22 (P ≤ 0.01).Conclusions: The findings supported the hypothesis that BgAIF plays a role in the adherence of B. glabrata hemocytes to sporocysts during schistosome infection in vitro. This demonstration of the activity of programmed gene editing will enable functional genomics approaches using CRISPR/Cas9 to investigate additional components of the snail-schistosome host-parasite relationship.

Diminished adherence of Biomphalaria glabrata embryonic cell line to sporocysts of Schistosoma mansoni following programmed knockout of the allograft inflammatory factor

Background: Larval development in an intermediate host gastropod snail of the genus Biomphalaria is an obligatory component of the life cycle of Schistosoma mansoni. Understanding of the mechanism(s) of host defense may hasten the development of tools that block transmission of schistosomiasis. The allograft inflammatory factor 1, AIF, which is evolutionarily conserved and expressed in phagocytes, is a marker of macrophage activation in both mammals and invertebrates. AIF enhances cell proliferation and migration. The embryonic cell line, termed Bge, from Biomphalaria glabrata is a versatile resource for investigation of the snail-schistosome relationship since Bge exhibits a hemocyte-like phenotype. Hemocytes perform central roles in innate and cellular immunity in gastropods and in some cases can kill the parasite. However, the Bge cells do not kill the parasite in vitro.Methods: Bge cells were transfected by electroporation with plasmid pCas-BgAIFx4, encoding the Cas9 nuclease and a guide RNA specific for exon 4 of the B. glabrata AIF (BgAIF) gene. Transcript levels for Cas9 and for BgAIF were monitored by quantitative reverse-transcription-PCR and, in parallel, adhesion of gene-edited Bge cells during co-culture with of schistosome sporocysts was assessed.Results: Gene knockout manipulation induced gene-disrupting indels, frequently 1-2 bp insertions and/or 8-30 bp deletions, at the programmed target site; a range from 9 to 17% of the copies of the BgAIF gene in the Bge population of cells were mutated. Transcript levels for BgAIF were reduced by up to 73% (49.5±20.2% S.D, P ≤ 0.05, n =12). Adherence by BgAIF gene-edited (ΔBgAIF) Bge to sporocysts diminished in comparison to wild type cells, although cell morphology did not change. Specifically, as scored by a semi-quantitative cell adherence index (CAI), fewer ΔBgAIF than control wild type cells adhered to sporocysts; control CAI, 2.66±0.10, ΔBgAIF, 2.30±0.22 (P ≤ 0.01).Conclusion: The findings supported the hypothesis that BgAIF plays a role in the adherence of B. glabrata hemocytes to sporocysts during schistosome infection in vitro. This demonstration of the activity of programmed gene editing will enable functional genomics approaches using CRISPR/Cas9 to investigate additional components of the snail-schistosome host-parasite relationship.

Diminished Adherence of Biomphalaria Glabrata Embryonic Cell Line to Sporocysts of Schistosoma Mansoni Following Programmed Knockout of the Allograft Inflammatory Factor

The development of the larval stage of Schistosoma mansoni in an intermediate host snail of the genus Biomphalaria is an obligatory component of the life cycle. Enhanced understanding of the mechanism(s) of host defense in the snail may hasten the development of tools that block transmission of schistosomiasis. The B. glabrata embryonic cell line, termed the Bge line, is a versatile resource for investigation of the snail-schistosome relationship. A key attribute of the Bge cell is the hemocyte-like phenotype, given the central role of the snail hemocyte in innate and cellular immunity. The allograft inflammatory factor 1, AIF, is evolutionarily conserved, typically is expressed in phagocytes and granular leukocytes, is a marker of macrophage activation in mammals and in invertebrates, and enhances cell proliferation and migration. AIF is highly expressed in strains of B. glabrata resistant S. mansoni infection in comparison with susceptible strains of the snail. We hypothesized that BgAIF may play a role in hemocyte proliferation, adhesion and/or migration after exposure of the snail to schistosomes. CRISPR/Cas gene knockout of the BgAIF gene in Bge cells was undertaken to investigate the hypothesis. Gene knockout manipulation induced gene-disrupting indels, frequently 1–2 bp insertions and/or 8–30 bp deletions, at the programmed target site; a range from 9 to 17% of BgAIF genes were mutated during 12 replicate experiments, and transcript levels for BgAIF were significantly reduced by up to 73% (range, 26 to 73%, mean 49.5 ± 20.2% S.D, n = 12) when monitored for up to nine days following the gene-editing manipulation. The adherence to sporocyst of BgAIF gene-edited (ΔBgAIF) Bge cells was significantly diminished in comparison to wild type cells, even though cell morphology did not differ between ΔBgAIF treatment and control groups of Bge cells. A Bge cell adherence index (CAI) to individual sporocysts was observed at 2.66 ± 0.10 for control and 2.30 ± 0.22 in ΔBgAIF cells (P < 0.05), revealing that ΔBgAIF cells were significantly less adherent than wild type Bge cells to primary sporocysts. The findings supported the hypothesis that BgAIF plays a role in the adherence of B. glabrata hemocytes to sporocysts during schistosome infection.

Diminished adherence of Biomphalaria glabrata embryonic cell line to sporocysts of Schistosoma mansoni following programmed knockout of the allograft inflammatory factor

BackgroundLarval development in an intermediate host gastropod snail of the genus Biomphalaria is an obligatory component of the life cycle of Schistosoma mansoni. Understanding of the mechanism(s) of host defense may hasten the development of tools that block transmission of schistosomiasis. The allograft inflammatory factor 1, AIF, which is evolutionarily conserved and expressed in phagocytes, is a marker of macrophage activation in both mammals and invertebrates. AIF enhances cell proliferation and migration. The embryonic cell line, termed Bge, from Biomphalaria glabrata is a versatile resource for investigation of the snail-schistosome relationship since Bge exhibits a hemocyte-like phenotype. Hemocytes perform central roles in innate and cellular immunity in gastropods and in some cases can kill the parasite. However, the Bge cells do not kill the parasite in vitro.MethodsBge cells were transfected by electroporation with plasmid pCas-BgAIFx4, encoding the Cas9 nuclease and a guide RNA specific for exon 4 of the B. glabrata AIF (BgAIF) gene. Transcript levels for Cas9 and for BgAIF were monitored by quantitative reverse-transcription-PCR and, in parallel, adhesion of gene-edited Bge cells during co-culture with of schistosome sporocysts was assessed.ResultsGene knockout manipulation induced gene-disrupting indels, frequently 1-2 bp insertions and/or 8-30 bp deletions, at the programmed target site; a range from 9 to 17% of the copies of the BgAIF gene in the Bge population of cells were mutated. Transcript levels for BgAIF were reduced by up to 73% (49.5±20.2% S.D, P ≤ 0.05, n =12). Adherence by BgAIF gene-edited (ΔBgAIF) Bge to sporocysts diminished in comparison to wild type cells, although cell morphology did not change. Specifically, as scored by a semi-quantitative cell adherence index (CAI), fewer ΔBgAIF than control wild type cells adhered to sporocysts; control CAI, 2.66±0.10, ΔBgAIF, 2.30±0.22 (P ≤ 0.01).ConclusionThe findings supported the hypothesis that BgAIF plays a role in the adherence of B. glabrata hemocytes to sporocysts during schistosome infection in vitro. This demonstration of the activity of programmed gene editing will enable functional genomics approaches using CRISPR/Cas9 to investigate additional components of the snail-schistosome host-parasite relationship.