pad track flag sirt1  (Addgene inc)

Journal: Scientific Reports

Article Title: Dynamics and necessity of SIRT1 for maternal–zygotic transition

doi: 10.1038/s41598-024-72595-6

Figure Lengend Snippet: Generation of the mouse Sirt1 loxPloxP ; Zp3 -Cre (cKO) model and reproductive trial of cKO females. ( A ) The PCR products indicate the recombined Sirt1 allele (the Sirt1 allele containing loxP-flanked exons 5–7) and Cre recombinase coding locus. ( B ) Quantitative RT‒PCR of Sirt1 cDNA in wt and Sirt1 null oocytes. The data are shown as the means ± SEMs of three independent replicates, each including at least 20 oocytes; significant differences were identified via unpaired t tests. ( C ) Histology of the ovaries of wt and cKO females; H&E staining. Scale bar: 500 µm. ( D ) Ovarian capacity is indicated by the ovarian reserve (i.e., the yield of GV oocytes per PMSG-treated female) and hormone responsiveness (i.e., IVO oocytes per PMSG-hCG-treated female). The results are shown as the means ± SEMs; the numbers of females are noted in brackets. ( E ) Oocyte mitochondrial abudance expressed by mtDNA copy number. Columns show mean ± SEMs of three independent experiments. Significant difference was tested using the unpaired t-test. ( F ) In the assessment of the reproductive phenotype, the conception rate was calculated as the number of matings per pregnancy; lines represent the median. The number of pups per litter (1st, 2nd, and 3rd) is shown as the mean ± SEM of three females. Wt: wild-type, cKO: conditional knockout, GV: germinal vesicle, IVO: in vivo ovulated.

Article Snippet: The SIRT1 coding sequence was cloned by PCR from pAd-Track Flag-SIRT1 (Addgene, #8438) into pYX-EYFP plasmid to create pYX-SIRT1-EYFP plasmid as described earlier . cRNA was produced via in vitro transcriptionusing the mMessage mMachineTM T3 Kit (Ambion, #1348) and was polyadenylated via the Poly(A) Tailing Kit (Ambion, #AM1350) according to the manufacturer’s protocols. cRNAs were purified using the RNeasy Mini Kit (Qiagen, #74,104) and stored at – 80 °C.

Techniques: Staining, Knock-Out, In Vivo

Journal: Scientific Reports

Article Title: Dynamics and necessity of SIRT1 for maternal–zygotic transition

doi: 10.1038/s41598-024-72595-6

Figure Lengend Snippet: The reappearance of SIRT1 in one-cell zygotes. ( A ) Immunocytochemical staining of SIRT1 in wt and Sirt1 null IVO oocytes. ( B ) SIRT1 is present in one-cell zygotes generated via parthenogenetic activation of wt and Sirt1 null oocytes. ( C ) Distribution and dynamics of SIRT1 during nuclear envelope breakdown (NEBD) in parthenogenetic zygotes; embryos expressing H2B‐mCHERRY (red) and SIRT1-EYFP (green) are shown. ( D ) Analysis of SIRT1 and H4K16ac in the male pronuclei of in vitro fertilized WT and Sirt1 null oocytes. The rectangle delimits the pronuclei (asterisk indicates the male pronucleus). Quantification of H4K16ac integrated density (IntDen) in pronuclei of wt and Sirt1 null zygotes and morphometry of IVF zygotes. Correlation analysis of H4K16ac with SIRT1 in wt zygotes. The Spearman coefficient is shown and was considered significant if P was ≤ 0.05 (bold). ( E ) Distribution and quantity of SIRT1 integrated density (IntDen) in wt parthenote zygotes after CHX treatment. ( F ) Overview of the relationship between the maternal SIRT1 protein and the Sirt1 transcript in the zygote (created in BioRender.com). The dots represent individual zygotes, and the lines represent the medians; significance was tested via the Mann‒Whitney U test (**, P < 0.01). IVO: in vivo ovulated; PA: parthenogenetic activation; NEBD: nuclear envelope breakdown; IVF: in vitro fertilization; VC: vehicle control; CHX: cycloheximide. Scale bar: 25 µm.

Article Snippet: The SIRT1 coding sequence was cloned by PCR from pAd-Track Flag-SIRT1 (Addgene, #8438) into pYX-EYFP plasmid to create pYX-SIRT1-EYFP plasmid as described earlier . cRNA was produced via in vitro transcriptionusing the mMessage mMachineTM T3 Kit (Ambion, #1348) and was polyadenylated via the Poly(A) Tailing Kit (Ambion, #AM1350) according to the manufacturer’s protocols. cRNAs were purified using the RNeasy Mini Kit (Qiagen, #74,104) and stored at – 80 °C.

Techniques: Staining, Generated, Activation Assay, Expressing, In Vitro, IF-P, In Vivo, Control

Journal: Scientific Reports

Article Title: Dynamics and necessity of SIRT1 for maternal–zygotic transition

doi: 10.1038/s41598-024-72595-6

Figure Lengend Snippet: Assessment of SIRT1 origin and maternal-to-embryonic SIRT1 exchange in two-cell (2C) embryos. ( A ) SIRT1 presence and quantification in two-cell parthenotes and IVF-produced embryos treated with α-amanitin (α-AM). ( B ) SIRT1 integrated density (IntDen) in two-cell (2C) IVF embryos; Sirt1 +/+ (wt) and Sirt1 +/- embryos were generated via fertilization of wt and Sirt1 null oocytes with wt sperm. ( C ) Summary of different sources of SIRT1 in two-cell wt embryos (created in BioRender.com). ( D ) Distribution and quantification of SIRT1 in pig four-cell (4C) and eight-cell (8C) parthenogenetic embryos treated with α-AM. ( E ) Interpretation of the findings obtained in mouse and porcine embryos after α-amanitin treatment (created from BioRender.com). Lines represent the median, and differences were evaluated via an unpaired Mann‒Whitney test (****, P < 0.0001). PN: pronuclear zygote; 2C/4C/8C: two-/four-/eight-cell embryo; PA: parthenogenetic activation; IVF: in vitro fertilization; VC: vehicle control; α-AM: α-amanitin; EGA: embryonic genome activation; ICC: immunocytochemistry. Scale bar: 50 µm.

Article Snippet: The SIRT1 coding sequence was cloned by PCR from pAd-Track Flag-SIRT1 (Addgene, #8438) into pYX-EYFP plasmid to create pYX-SIRT1-EYFP plasmid as described earlier . cRNA was produced via in vitro transcriptionusing the mMessage mMachineTM T3 Kit (Ambion, #1348) and was polyadenylated via the Poly(A) Tailing Kit (Ambion, #AM1350) according to the manufacturer’s protocols. cRNAs were purified using the RNeasy Mini Kit (Qiagen, #74,104) and stored at – 80 °C.

Techniques: Produced, Generated, Activation Assay, In Vitro, Control, Immunocytochemistry

Journal: Scientific Reports

Article Title: Dynamics and necessity of SIRT1 for maternal–zygotic transition

doi: 10.1038/s41598-024-72595-6

Figure Lengend Snippet: Developmental competence of Sirt1 +/- embryos and inhibition of SIRT1 in post-EGA embryos. ( A ) IVF output and blastocyst quality. In vitro fertilization of wt and Sirt1 null oocytes with wt spermatozoa generated the Sirt1 +/+ and Sirt1 +/- genotypes, respectively. The columns represent the means ± SEMs of the fertilization rate, cleavage rate, blastocyst rate, and hatching rate in the five IVF assays; differences were evaluated via paired t tests. ( B ) SIRT1 localization in IVF blastocysts. The dots represent the SIRT1 integrated density (IntDen) of individual blastocysts, and the lines represent the median; differences were evaluated via the Mann‒Whitney U test (****, P < 0.0001). Scale bar: 50 µm. ( C ) Scheme of SIRT1 inhibition in post-EGA (wt) embryos subjected to in vitro culture of in - vivo vivo-produced post-EGA (2C) embryos. Sirtinol was used as a selective SIRT1 inhibitor during embryo culture. ( D ) Embryonic development of embryos treated with vehicle (0.1% DMSO) or sirtinol (10 µM). The columns represent the means ± SEMs of the blastocyst rates and hatching rates from three independent assays; differences were evaluated via paired t tests. Scale bar: 500 µm. ( E ) SIRT1 and H4K16ac colocalization in sirtinol-treated blastocysts. The dots represent the integrated density (IntDen) of H4K16ac and SIRT1 in individual blastocysts, and the lines represent the median; differences were evaluated via the Mann‒Whitney U test (****, P < 0.0001). Scale bar: 50 µm.

Article Snippet: The SIRT1 coding sequence was cloned by PCR from pAd-Track Flag-SIRT1 (Addgene, #8438) into pYX-EYFP plasmid to create pYX-SIRT1-EYFP plasmid as described earlier . cRNA was produced via in vitro transcriptionusing the mMessage mMachineTM T3 Kit (Ambion, #1348) and was polyadenylated via the Poly(A) Tailing Kit (Ambion, #AM1350) according to the manufacturer’s protocols. cRNAs were purified using the RNeasy Mini Kit (Qiagen, #74,104) and stored at – 80 °C.

Techniques: Inhibition, In Vitro, Generated, In Vivo, Produced, Embryo Culture

Journal: Scientific Reports

Article Title: Dynamics and necessity of SIRT1 for maternal–zygotic transition

doi: 10.1038/s41598-024-72595-6

Figure Lengend Snippet: Analysis of the SIRT1-H4K16ac interaction in mouse and human blastocysts. ( A ) SIRT1 and H4K16ac localization in in vivo-produced blastocysts produced via mating wt and cKO females with wt males. The dots represent the H4K16ac integrated density (IntDen) of individual blastocysts, and the lines represent the median; differences were evaluated via the Mann‒Whitney U test (**, P < 0.01; ***, P < 0.001). ( B ) Correlation of blastocyst parameters and regression analysis of H4K16ac-blastomere counts. ( C ) Maximum intensity projection of Z-stacks of SIRT1 and H4K16ac in human blastocysts. Three-dimensional (3D) regions of interest (ROIs) of individual blastomeres defined by the DAPI signal. Correlation analysis of the sum intensity and number of cells in the blastocyst. ( D ) Co-localization analysis of SIRT1 and H4K16ac in blastomeres. The image represents the analysed blastocysts, and the rectangle highlights the cells used for H4K16ac and SIRT1 colocalization analysis. The number ( N ) of analysed cells of 13 blastocysts is indicated. ( E ) Linear regression and correlation analysis of the mean intensity of SIRT1 and H4K16ac in the subpopulation of human blastomeres. Maximum intensity projection was used, and the rectangle shows an example of an ROI for the analysed blastomeres (10 blastomeres per blastocyst). The Spearman coefficient was calculated, and P values are noted.

Article Snippet: The SIRT1 coding sequence was cloned by PCR from pAd-Track Flag-SIRT1 (Addgene, #8438) into pYX-EYFP plasmid to create pYX-SIRT1-EYFP plasmid as described earlier . cRNA was produced via in vitro transcriptionusing the mMessage mMachineTM T3 Kit (Ambion, #1348) and was polyadenylated via the Poly(A) Tailing Kit (Ambion, #AM1350) according to the manufacturer’s protocols. cRNAs were purified using the RNeasy Mini Kit (Qiagen, #74,104) and stored at – 80 °C.

Techniques: In Vivo, Produced

Journal: Scientific Reports

Article Title: Dynamics and necessity of SIRT1 for maternal–zygotic transition

doi: 10.1038/s41598-024-72595-6

Figure Lengend Snippet: Generation of the Sirt1 -/- genotype, embryonic development and transcriptomic analysis of Sirt1 null embryos. ( A ) Number of pups per litter after mating with a Sirt1 +/- male. The dots represent individual litters, and the lines represent the medians; significance was tested via the Mann‒Whitney U test (**, P < 0.01). Ratio of offspring genotypes following mating with Sirt1 +/- males. Stacked columns show the cumulative proportion of recorded genotypes. A one sample t test was used to compare the recorded genotype ratio with a hypothetical value (i.e., 0.5). ( B ) Live-cell imaging and time analysis of early embryonic development of the Sirt1 +/+ and Sirt1 -/- genotypes following the activation of wt and Sirt1 null oocytes, respectively (red: H2B‐tdTomato). Assessment of cleavage (24 h) and blastocyst rates (96 h). The columns represent the means ± SEMs of three independent IVF assays, and significance was evaluated via paired t tests (**, P < 0.01). ( C ) Zygote development and viability. Stacked columns show the cumulative proportions of recorded phenotypes of zygotes; significance was evaluated via the chi-square test (***, P < 0.001). ( D ) Design of two schemes of nascent mRNA analysis using 5-ethynyl uridine (5-EU) treatment. ( E ) Representative images and quantification of 5-EU incorporated into nascent mRNA in parthenogenetically activated wt and Sirt1 null oocytes. ( F ) Immunocytochemical staining and analysis of H4K16ac in two-cell parthenote embryos. Dot plots show individual values (i.e., embryos) of integrated density (IntDen), and the lines represent the median; significance was evaluated via the Mann‒Whitney U test. ( G ) Heatmap of transcripts expressed in bulk samples of wt and Sirt1 null parthenote embryos. (H) Qualitative RNA analysis and volcano plot of the RNA-seq data. FDR = false discovery rate; |LFC|= absolute value of log 2 fold change. Scale bar: 50 µm.

Article Snippet: The SIRT1 coding sequence was cloned by PCR from pAd-Track Flag-SIRT1 (Addgene, #8438) into pYX-EYFP plasmid to create pYX-SIRT1-EYFP plasmid as described earlier . cRNA was produced via in vitro transcriptionusing the mMessage mMachineTM T3 Kit (Ambion, #1348) and was polyadenylated via the Poly(A) Tailing Kit (Ambion, #AM1350) according to the manufacturer’s protocols. cRNAs were purified using the RNeasy Mini Kit (Qiagen, #74,104) and stored at – 80 °C.

Techniques: Live Cell Imaging, Activation Assay, Staining, RNA Sequencing