p2a egfp sequence  (Addgene inc)

Journal: bioRxiv

Article Title: Independent evolution of holocentric centromeres in an early branching apicomplexan parasite

doi: 10.1101/2025.09.30.679541

Figure Lengend Snippet: (A) Phylogenetic relationship of C. parvum H3 and centromeric H3 (CENH3) histones and their corresponding top BLASTP hits in other eukaryotes, apicomplexan parasites, Saccharomyces cerevisiae , or Homo sapiens . Cyan stars represent annotated histone H3 genes. (B) Protein sequence alignment of H3 (cyan) and CENH3 (black) in C. parvum . Amino acids that are identical between at least two sequences are highlighted in grey. (C) Diagram of the targeting constructs designed to replace the endogenous tk locus with a second copy of either of the genes annotated as Histone H3-like proteins in C. parvum ( cgd3_2540 or cgd4_3220 ), a 3HA tag, and a Nluc-P2A-NeoR cassette. (D) Immunofluorescence staining of transgenic H3.1-3HA or (E) H3.2-3HA parasites. HCT-8 cells were infected with transgenic oocysts, fixed at 18 hpi, and stained with rat anti-HA and VVL-Biotin, followed by secondary antibodies Alexa Fluor 488 goat anti-rat IgG and Alexa Fluor 647 Streptavidin. Hoechst was used for nuclear staining. Images were acquired as Z-stacks using a Zeiss LSM-880 Laser Scanning Confocal microscope equipped with Airyscan (LSCM-A) and are presented with orthogonal views. Scale bars, 1 μm.

Article Snippet: 2) The 3HA-pLinker region was amplified from a TK-3HA-CENH3-Nluc-P2A-Neo R -TK plasmid (data not shown in this study), 3) the GBP CDS was amplified from C. parvum genomic DNA, 4) the pLinker-3Ty sequence was amplified from a tagging plasmid previously generated in our lab [ ], and 5) the P2A skip peptide sequence was generated as a gBlock Gene Fragment from Azenta.

Techniques: Sequencing, Construct, Immunofluorescence, Staining, Transgenic Assay, Infection, Microscopy

Journal: bioRxiv

Article Title: Independent evolution of holocentric centromeres in an early branching apicomplexan parasite

doi: 10.1101/2025.09.30.679541

Figure Lengend Snippet: (A) Diagram of the targeting construct designed to add a 3HA tag and Nluc-P2A-Neo R cassette to the C-terminus of the C. parvum CENH3 ( cgd4_2030 ). (B) HCT-8 cells were infected with CENH3-3HA C. parvum parasites, fixed at 22 hpi, and stained with rat anti-HA and VVL-Biotin, followed by secondary antibodies Alexa Fluor 488 goat anti-rat IgG and Alexa Fluor 647 Streptavidin followed by Hoechst staining. Images were acquired as Z-stacks using LSCM-A and are presented with orthogonal views. Scale bars, 1 μm. (C) Cartoon depiction corresponding to the microscopy image in panel (B). In Cryptosporidium , a late-stage meront contains eight nuclei that have recently completed cytokinesis to form mature merozoites. The CENH3-3HA signal within each nucleus displays a diffuse staining pattern in C. parvum . (D) HCT-8 cells were infected with CENH3-3HA T. gondii parasites, fixed at 24 hours post-infection (hpi), and stained with rat anti-HA and rabbit anti-aldolase (ALD) to visualize the cytosol, followed by secondary antibodies Alexa Fluor 488 goat anti-rat IgG and Alexa Fluor 568 anti-rabbit IgG. Hoechst was used for nuclear staining. Images were acquired as Z-stacks using LSCM-A and are presented with orthogonal views. Scale bars, 1 μm. (E) Cartoon depiction corresponding to the microscopy image in panel (D). In T. gondii tachyzoites, the CENH3-3HA signal within each nucleus displays a discrete staining pattern. (F) HCT-8 cells were infected with excysted C. parvum sporozoites for 2 h, then washed twice to remove extracellular parasites. Cultures were fixed at 30 min increments between 6-9 hpi to identify the first (trophozoites with one nucleus), second (early meronts with 2 nuclei), and third (mid-stage meronts with 4 nuclei) mitotic divisions during the first round of merogony by widefield microscopy. Scale bars, 1 μm. (G) Cultures were infected with CENH3-3HA C. parvum sporozoites for 2 h, washed, and fixed at 6.5 hpi to capture the first round of mitosis. Cultures were stained with rat anti-HA, rabbit anti-centrin-1, and VVL-Biotin, followed by secondary antibodies Alexa Fluor 488 goat anti-rat IgG, Alexa Fluor 568 goat anti-mouse IgG, and Alexa Fluor 647 Streptavidin, and lastly Hoechst. Parasites undergoing mitosis were identified by having two centrin-1 points per nucleus, whereas parasites in interphase had only one centrin-1 point. Images were acquired as Z-stacks using LSCM-A and are presented with orthogonal views. Scale bars, 1 μm.

Article Snippet: 2) The 3HA-pLinker region was amplified from a TK-3HA-CENH3-Nluc-P2A-Neo R -TK plasmid (data not shown in this study), 3) the GBP CDS was amplified from C. parvum genomic DNA, 4) the pLinker-3Ty sequence was amplified from a tagging plasmid previously generated in our lab [ ], and 5) the P2A skip peptide sequence was generated as a gBlock Gene Fragment from Azenta.

Techniques: Construct, Infection, Staining, Microscopy

Journal: bioRxiv

Article Title: Independent evolution of holocentric centromeres in an early branching apicomplexan parasite

doi: 10.1101/2025.09.30.679541

Figure Lengend Snippet: (A) The 3’ overhang single-stranded DNA at the end of telomeres contains repetitive sequences (GGTTTA) n in C. parvum that form G-quadruplex structures. (B) The G-quadruplex binding protein (GBP) is essential for forming and stabilizing these structures. The sequence of Cp GBP and its binding motif has been previously determined and the protein structure is modeled by Alphafold and ChimeraX . (C) Diagram of the targeting construct designed to add a 3HA tag and Nluc-P2A-Neo R cassette to the C-terminus of the G-quadruplex binding proteins gene ( GBP , cgd1_3530) (D) HCT-8 cells were infected with GBP-3HA parasites, fixed at 6.5 hpi to capture early meronts (2 nuclei), and stained with rat anti-HA and VVL-Biotin, followed by secondary antibodies Alexa Fluor 488 goat anti-rat IgG and Alexa Fluor 647 Streptavidin. Hoechst was used for nuclear staining. Images were acquired as Z-stacks using LSCM-A and are presented with orthogonal views and the scale bar is 1 μm. (E) Diagram of the targeting construct designed to replace the endogenous tk locus with a second copy of CENH3 N-terminally tagged with 3HA and GBP with a C-terminal 3xTy tag separated with a P2A split peptide. (F) Immunofluorescence staining of transgenic 3HA-CENH3-GBP-3Ty parasites. HCT-8 cells were infected with transgenic oocysts, fixed at 6.5 hpi to capture early meronts (2 nuclei), and stained with rat anti-HA, mouse anti-Ty, and VVL-Biotin followed by a secondary antibody stain containing Alexa Fluor 488 goat anti-rat IgG, Alexa Fluor 568 goat-anti mouse, and Alexa Fluor 647 Streptavidin. Hoechst was used for nuclear staining. Images were acquired as Z-stacks using LSCM-A and are presented with orthogonal views. Scale bars, 1 μm.

Article Snippet: 2) The 3HA-pLinker region was amplified from a TK-3HA-CENH3-Nluc-P2A-Neo R -TK plasmid (data not shown in this study), 3) the GBP CDS was amplified from C. parvum genomic DNA, 4) the pLinker-3Ty sequence was amplified from a tagging plasmid previously generated in our lab [ ], and 5) the P2A skip peptide sequence was generated as a gBlock Gene Fragment from Azenta.

Techniques: Binding Assay, Sequencing, Construct, Infection, Staining, Immunofluorescence, Transgenic Assay

Journal: iScience

Article Title: Aurora B and INCENP co-overexpression severely disrupts mitosis and distinctly modifies the global transcriptional landscape.

doi: 10.1016/j.isci.2025.112731

Figure Lengend Snippet: Figure 1. Concurrent overexpression of AurkB and INCENP promotes a synergistic increase in Aurora B kinase activity (A and B) Human RPE-1 cells were transduced with lentiviruses carrying vectors for overexpression of AurkB, INCENP or both proteins (AurkB+INCENP). Western blots show intracellular levels of AurkB and INCENP protein 72 h post-lentiviral infection (A) and in cells that, after transduction, were cultured for an extended period and then FACS-sorted to isolate stable cell lines (B). Total H3 and H3S10P amounts are displayed as an indicator of AurkB activity. Actin levels were used as a loading control. (C–G) RPE-1 cells carrying an extra copy of AURKB (AurkB), INCENP (INCENP) or a bicistronic construct that comprises both genes linked by a sequence encoding a P2A ‘‘self-cleaving’’ oligopeptide (AurkB+INCENP), all under the control of the TET promoter, were alternatively used to conditionally overexpress AurkB and/or INCENP. (C) Western blot shows total AurkB, INCENP, H3, H3S10P and actin protein levels 32 h after addition of 2 μg/mL tetracycline. (D–G) Immunofluorescence pictures showing AurkB (D–F) or Survivin (G) (both in green), as well as INCENP (in red) localization. DAPI (in blue) and merged images are also provided. Scale bars, 10 μm. (D-F) AurkB and INCENP distribution was evaluated at different cell cycle stages from pro-metaphase to telophase in control cells (D) and in cells that overexpressed AurkB and INCENP at moderately low (overexpression) (E) or at high levels (high overexpression) (F). In the latter case, Survivin localization was also analyzed (G).

Article Snippet: Likewise, to achieve their simultaneous overexpression, both the INCENP and AURKB ORFs, separated by the P2A sequence from pCW57-MCS1-2A-MCS2 (Addgene), were introduced in the pCDNA5/FRT/TO plasmid under control of the TET promoter.

Techniques: Over Expression, Activity Assay, Transduction, Western Blot, Infection, Cell Culture, Stable Transfection, Control, Construct, Sequencing, Immunofluorescence

Journal: iScience

Article Title: Aurora B and INCENP co-overexpression severely disrupts mitosis and distinctly modifies the global transcriptional landscape.

doi: 10.1016/j.isci.2025.112731

Figure Lengend Snippet: Figure 4. H3S10P is enriched genome-wide after the concurrent increase in AurkB and INCENP protein levels (A–F) RPE-1 cells carrying an extra copy of AURKB (AurkB), INCENP (INCENP) or a bicistronic construct that comprises both genes linked by a sequence encoding a P2A ‘‘self-cleaving’’ oligopeptide (AurkB+INCENP, A + I), all under the control of the TET promoter, were used to evaluate H3S10P distribution 32 h after addition of 2 μg/mL tetracycline. (A) Immunofluorescence images display the sub-cellular distribution of H3S10P (in green) in selected cells, both during interphase and in metaphase. DAPI (in blue) and a merged image (Merged) are also included. Scale bars, 10 μm. (B) Graph shows the percentages of interphase cells presenting (+) or not (−) positive H3S10P signal, both in interphase (Inter) and in metaphase (Meta). Overexpressed AurkB and/or INCENP proteins were

Article Snippet: Likewise, to achieve their simultaneous overexpression, both the INCENP and AURKB ORFs, separated by the P2A sequence from pCW57-MCS1-2A-MCS2 (Addgene), were introduced in the pCDNA5/FRT/TO plasmid under control of the TET promoter.

Techniques: Genome Wide, Construct, Sequencing, Control, Immunofluorescence