bub1b coding sequence cds  (Addgene inc)

Journal: bioRxiv

Article Title: Molecular Contribution to Embryonic Aneuploidy and Genotypic Complexity During Initial Cleavage Divisions of Mammalian Development

doi: 10.1101/2020.07.24.220475

Figure Lengend Snippet: (A) In vitro produced bovine oocytes underwent IVF and the resulting zygotes non-invasively monitored by time-lapse image analysis until collection for immunostaining of nuclear structure. Another subset of zygotes was microinjected with fluorescently labeled modified mRNAs and chromosome segregation visualized during the first three mitotic divisions in real-time by live-cell confocal microscopy. Cleavage-stage embryos were disassembled into single blastomeres at the 2- to 12-cell stage for scDNA-seq and CNV analysis to determine the precise frequency of aneuploidy at multiple cleavage stages. Other zygotes were microinjected with non-overlapping morpholinos targeting the mitotic checkpoint protein, BUB1B, and/or modified BUB1B mRNA to test the effect and specificity of MCC inhibition on chromosome segregation, division dynamics, and preimplantation development. Gene expression profiling was also conducted on a subset of MCC deficit zygotes versus controls by quantitative RT-PCR to identify changes in gene abundance and molecular pathways associated with BUB1B knockdown. (B) Immunostaining of zygotes and (C) cleavage-stage embryos with LMNB1 (green) using DAPI (blue) to visualize DNA revealed several micro- and multi-nuclei (white arrows). (D) Blastocysts also immunostained for the trophoblast marker, CDX2 (red), showed that micronuclei are often present in the TE, (E) but can also be retained within the ICM of the embryo. Scale bar = 10μm.

Article Snippet: The BUB1B coding sequence (CDS) was amplified from the plasmid, pcDNA5-EGFP-AID-BubR1 (Addgene #47330), followed by mutation of the MAO binding site using the Q5 site directed mutagenesis kit (NEB) according to the manufacturer’s instructions.

Techniques: In Vitro, Produced, Immunostaining, Labeling, Modification, Confocal Microscopy, Inhibition, Gene Expression, Quantitative RT-PCR, Knockdown, Marker

Journal: bioRxiv

Article Title: Molecular Contribution to Embryonic Aneuploidy and Genotypic Complexity During Initial Cleavage Divisions of Mammalian Development

doi: 10.1101/2020.07.24.220475

Figure Lengend Snippet: (A) DNA sequences of two non-overlapping MAOs designed to target the ATG start site (shown in red, BUB1B MAO #1) and the 5’ UTR (depicted in blue, BUB1B MAO #2) of BUB1B. (B) BUB1B knockdown efficiency was assessed in synchronized MDBK cells following 48 hours of treatment with 3μl/ml of colcemid alone (non-transfected), the Std control MAO, or BUB1B MAO #1 via immunofluorescence. BUB1B protein expression was analyzed in DAPI stained (blue) MDBK cells. Note the lack of or reduced number of BUB1B positive foci (red) in the BUB1B MAO #1 treated cells compared to the controls; Scale bar = 20μm. (C) Bar graph showing the percentage of MDBK cells in metaphase with BUB1B expression after colcemid treatment (black) or transfection with different concentrations (2, 4, and 8 μM) of the Std control MAO (blue) or BUB1B MAO #1 (red). While the number of cells exhibiting BUB1B positive foci was similar between the non-transfected and Std MAO controls, a dose-dependent decrease in BUB1B expression was observed following BUB1B MAO #1 treatment.

Article Snippet: The BUB1B coding sequence (CDS) was amplified from the plasmid, pcDNA5-EGFP-AID-BubR1 (Addgene #47330), followed by mutation of the MAO binding site using the Q5 site directed mutagenesis kit (NEB) according to the manufacturer’s instructions.

Techniques: Knockdown, Transfection, Control, Immunofluorescence, Expressing, Staining

Journal: bioRxiv

Article Title: Molecular Contribution to Embryonic Aneuploidy and Genotypic Complexity During Initial Cleavage Divisions of Mammalian Development

doi: 10.1101/2020.07.24.220475

Figure Lengend Snippet:

Article Snippet: The BUB1B coding sequence (CDS) was amplified from the plasmid, pcDNA5-EGFP-AID-BubR1 (Addgene #47330), followed by mutation of the MAO binding site using the Q5 site directed mutagenesis kit (NEB) according to the manufacturer’s instructions.

Techniques: Microinjection, Control

Journal: bioRxiv

Article Title: Molecular Contribution to Embryonic Aneuploidy and Genotypic Complexity During Initial Cleavage Divisions of Mammalian Development

doi: 10.1101/2020.07.24.220475

Figure Lengend Snippet: (A) Darkfield time-lapse imaging frames depicting the various embryo phenotypes (red arrows), including attempted division, multipolar division, and blastomere asymmetry observed following BUB1B MAO #1 or (B) BUB1B MAO #2 microinjection in bovine zygotes. (C) Representative stereomicroscope images of embryos and blastocysts from the Std control MAO, BUB1B MAO #1, and BUB1B MAO #1 plus BUB1B modified mRNA treatment groups. (D) Bar graph of the percentage of embryos that reached the blastocyst stage in non-injected, Std control MAO, BUB1B MAO #1, BUB1B MAO #2, or BUB1B MAO #1 plus BUB1B modified mRNA injected zygotes. While no blastocysts were obtained following BUB1B MAO #1 or #2 treatment, the co-injection of BUB1 MAO #1 and BUB1B modified mRNA was able to almost fully rescue the phenotype and restore blastocyst formation rates to that observed in controls. (E) Confocal images of LMNB1 (green) immunostaining in BUB1B MAO #1 or #2 treated embryos stained with DAPI (blue). Severely abnormal nuclear morphology and the presence of both micro- and multi-nuclei were detected (denoted with white arrowheads) in embryos at the zygote stage (top row) and cleavage-stage that exhibited abnormal cell divisions (bottom row). Note the DNA without nuclear envelope (white arrows) and the blastomere that completely lacked nuclear material in the 2-cell embryo located in the lower left image; Scale bars = 10μm. (E) CNV plots of blastomeres from different cleavage-stage embryos disassembled into single cells following BUB1B #1 MAO injection. While some euploid blastomeres were detected in BUB1B-injected embryos (upper left plot), most exhibited chaotic aneuploidy with multiple whole and sub-chromosomal losses and gains.

Article Snippet: The BUB1B coding sequence (CDS) was amplified from the plasmid, pcDNA5-EGFP-AID-BubR1 (Addgene #47330), followed by mutation of the MAO binding site using the Q5 site directed mutagenesis kit (NEB) according to the manufacturer’s instructions.

Techniques: Imaging, Microinjection, Control, Modification, Injection, Immunostaining, Staining

Journal: bioRxiv

Article Title: Molecular Contribution to Embryonic Aneuploidy and Genotypic Complexity During Initial Cleavage Divisions of Mammalian Development

doi: 10.1101/2020.07.24.220475

Figure Lengend Snippet: Heat map of all mitotic, cell cycle, developmentally-regulated, and cell survival genes assessed in individual BUB1B MAO #1 versus non-injected and Std Control-injected MAO bovine zygotes via microfluidic qRT-PCR. Cycle threshold (Ct) values were normalized to the most stable reference genes (RPL15 and GUSB) across embryo groups and presented as the average. Gray squares indicated no expression, whereas yellow, white, and purple squares correspond to low, medium, and high expression, respectively.

Article Snippet: The BUB1B coding sequence (CDS) was amplified from the plasmid, pcDNA5-EGFP-AID-BubR1 (Addgene #47330), followed by mutation of the MAO binding site using the Q5 site directed mutagenesis kit (NEB) according to the manufacturer’s instructions.

Techniques: Injection, Control, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Molecular Contribution to Embryonic Aneuploidy and Genotypic Complexity During Initial Cleavage Divisions of Mammalian Development

doi: 10.1101/2020.07.24.220475

Figure Lengend Snippet: The relative abundance of several mitotic, cell cycle, developmentally-regulated, and cell survival genes was assessed via microfluidic quantitative RT-PCR (qRT-PCR) in non-injected (NI; N=5), Std Control MAO (N=5), and BUB1B MAO #1 (N=5) individual zygotes using gene-specific primers. (A) The genes that were significantly downregulated (p<0.05) in BUB1B MAO-injected embryos compared to the NI and Std Control MAO groups + standard error is shown in the bar graph. (B) A bar graph of the genes that were significantly upregulated in BUB1B MAO-injected embryos relative to the controls + standard error. CNRQ values of each gene was compared across embryo groups using the Mann-Whitney U-test. The full list of the 96 genes with primer sequences assessed by qRT-PCR is available in and Supplemental Table S2 , respectively.

Article Snippet: The BUB1B coding sequence (CDS) was amplified from the plasmid, pcDNA5-EGFP-AID-BubR1 (Addgene #47330), followed by mutation of the MAO binding site using the Q5 site directed mutagenesis kit (NEB) according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Injection, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Molecular Contribution to Embryonic Aneuploidy and Genotypic Complexity During Initial Cleavage Divisions of Mammalian Development

doi: 10.1101/2020.07.24.220475

Figure Lengend Snippet: (A) Simplified model of how the lack of maternal and paternal pronuclear fusion (syngamy) at the zygote stage, followed by genome duplication and multipolar divisions, contributes to blastomeres with uniparental origins, or those that only contain maternal or paternal DNA. (B) Live-cell imaging also revealed the formation of anaphase lagging chromosomes likely from merotelic attachments prior to or during the first mitotic division. The chromosome(s) become encapsulated in nuclear envelope to form a micronucleus and the embryo continues to divide normally. In these subsequent bipolar divisions, most micronuclei either fuse back with the primary nucleus upon nuclear envelope breakdown or persist and undergo unilateral inheritance, but some micronuclei form a chromatin bridge with the nucleus of another blastomere during anaphase. (C) The depletion of BUB1B in zygotes resulted in no division or attempted division and embryo arrest, while multipolar divisions, blastomere asymmetry, and micro-/multi-nuclei were observed in MCC-deficient embryos that completed the first cytokinesis. These abnormal divisions also produced daughter cells with chaotic aneuploidy and/or empty blastomeres with no nuclear structure that induced embryo arrest and suggested that the lack of MCC permits the genotypic complexity detected at the early cleavage-stages of preimplantation development.

Article Snippet: The BUB1B coding sequence (CDS) was amplified from the plasmid, pcDNA5-EGFP-AID-BubR1 (Addgene #47330), followed by mutation of the MAO binding site using the Q5 site directed mutagenesis kit (NEB) according to the manufacturer’s instructions.

Techniques: Live Cell Imaging, Produced