Journal: bioRxiv

Article Title: A genome-wide knock-out screen for actors of epigenetic silencing reveals new regulators of germline genes and 2-cell like cell state

doi: 10.1101/2021.05.03.442415

Figure Lengend Snippet: (A) mScarlet expression in the 6 indicated KOs, rescued with their respective V5-tagged protein-coding sequence (CDS), or with an empty vector. Each data point is an individual clone (n=3 clones per KO). (B) Hygromycin resistance after rescue. One representative clone is shown for each KO. (C) Dazl expression after rescue (n=3 rescued clones per KO). (D) Western blot analysis of DAZL and the indicated V5-tagged CDS. (E) RT-qPCR assays showing the expression changes of Dazl , differentiation ( Fgf5 ) and pluripotency ( Prdm14, Pou5f1 ) markers upon spontaneous differentiation after withdrawal of LIF. (F) MeDIP assay showing the relative levels of 5mC at the Dazl promoter for 6 candidates KO, compared to the parental DASH mES cells (n=3 clones per KO). (G) LUMA assay showing the changes in the global level of DNA methylation for 6 candidates KO, compared to the parental DASH mES cells (n=3 clones per KO). (H and I) LC-MS/MS assay showing the changes in the global level of 5mdC/C (H) and 5hmdC/C (I) for 6 candidates KO, compared to the parental DASH mES cells (n=3 clones per KO).

Article Snippet: For rescue experiments, the coding sequence (CDS) of candidates was synthesized (for Bend3, Spop, and Zbtb14) (GenScript), amplified from cDNA ( Kdm5c ), or obtained from colleagues (for Mcm3ap , kindly shared by Prof. N. Sakaguchi, ).

Techniques: Expressing, Sequencing, Plasmid Preparation, Clone Assay, Western Blot, Quantitative RT-PCR, Methylated DNA Immunoprecipitation, DNA Methylation Assay, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: A genome-wide knock-out screen for actors of epigenetic silencing reveals new regulators of germline genes and 2-cell like cell state

doi: 10.1101/2021.05.03.442415

Figure Lengend Snippet: (A) RT-qPCR analysis of the expression of 2C-specific genes in Spop KO, Mcm3ap KO, Kdm5c KO, and Zbtb14 KO, compared to the parental DASH mES cells (n=3 clones per KO). (B) Western blot analysis of ZSCAN4 and MERVL-Gag for Spop KO, Mcm3ap KO, Kdm5c KO, Zbtb14 KO, and parental DASH mES cells cultured in Serum or 2i. (C) RT-qPCR analysis of the expression of 2C-specific genes for Spop KO, Mcm3ap KO, Kdm5c KO, and Zbtb14 KO, transfected with either an empty plasmid or their respective CDS, compared to the parental DASH mES cells (n=3, clones per KO). (D) Western blot analysis of V5, ZSCAN4, and MERVL-Gag in 4 candidates KO, transfected with an empty plasmid or their respective V5-tagged CDS. (E) Model for role of novel epigenetic factors ZBTB14, KDM5C, SPOP, MCM3AP, BEND3, and KMT2D in the regulation of germline genes, and 2 cell-like cell state.

Article Snippet: For rescue experiments, the coding sequence (CDS) of candidates was synthesized (for Bend3, Spop, and Zbtb14) (GenScript), amplified from cDNA ( Kdm5c ), or obtained from colleagues (for Mcm3ap , kindly shared by Prof. N. Sakaguchi, ).

Techniques: Quantitative RT-PCR, Expressing, Clone Assay, Western Blot, Cell Culture, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: A genome-wide knock-out screen for actors of epigenetic silencing reveals new regulators of germline genes and 2-cell like cell state

doi: 10.1101/2021.05.03.442415

Figure Lengend Snippet: (A) RT-qPCR analysis of the expression of 2C-specific genes in DASH mES cells cultured in 2i, Bend3 KO, and Kmt2d KO. (B) Western blot analysis of ZSCAN4 and MERVL-Gag for Bend3 KO, Kmt2d KO, and parental DASH mES cells cultured in Serum or 2i. (C) RT-qPCR analysis of the expression of 2C-specific genes for Bend3 KO (n=3, clones), transfected with an empty plasmid or their respective CDS, compared to the parental DASH mES cells. (D) Western blot analysis of ZSCAN4 and MERVL-Gag for Kdm5c KO clones, transfected with an empty plasmid, or a wild-type Kdm5c CDS, or a catalytically dead Kdm5c CDS (H514A). (E) RT-qPCR analysis of the expression of 2C-specific genes for Kdm5c KO clones, transfected with an empty plasmid, or a wild-type Kdm5c CDS, or a catalytically dead Kdm5c CDS (H514A).

Article Snippet: For rescue experiments, the coding sequence (CDS) of candidates was synthesized (for Bend3, Spop, and Zbtb14) (GenScript), amplified from cDNA ( Kdm5c ), or obtained from colleagues (for Mcm3ap , kindly shared by Prof. N. Sakaguchi, ).

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Western Blot, Clone Assay, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Prion infection, transmission and cytopathology modelled in a low-biohazard human cell line

doi: 10.1101/2020.04.01.019786

Figure Lengend Snippet: (A ) Generation of SH-SY5Y Δ PRNP cell lines expressing the ovine VRQ PrP C variant and its subsequent infection with the PG127 strain of sheep-derived prions passaged in tg338 mice. Hin dIII and Eco RI restriction sites were used to clone the ovine PRNP construct. Ticks in the plasmid map correspond to increments of 1000 base pairs. hSP = human signal peptide (purple), oSP = ovine signal peptide (blue), hovS = monoclonal ovSH-SY5Y, povS = polyclonal ovSH-SY5Y, ovSH-SY5Y = SH-SY5Y Δ PRNP transfected with a plasmid harboring the sequence for ovine PRNP (ov PRNP ). (B) Western blot analysis comparing the expression levels of PrP C in hovS and povS with those in wt SH-SY5Y and in the human cell lines U251-MG and LN229. HovS and povS showed similar PrP C expression levels as U251-MG and LN229, whereas the levels in wt SH-SY5Y were slightly lower. SH-SY5Y Δ PRNP cells were used as negative control and actin as loading control. The anti-PrP antibody POM2 was used for detection. (C) Confocal imaging to detect cell surface exposed PrP C on hovS and povS. hovS and a subpopulation of povS showed a strong signal for cell surface exposed PrP C , whereas no detectable signal was visible for SH-SY5Y Δ PRNP . LN229 cells were used as positive control. The anti-PrP antibody POM1 (here and henceforth) was used for detection of PrP.

Article Snippet: PRNP coding sequence (CDS, 774 bp) of Ovis aries harboring the VRQ allele (GeneScript, Piscataway Townsip, NJ, USA), codon optimized for expression in human cell lines and modified to include the human ER localization signal was cloned into the expression vector (Catalog Nr: OGS606-5U) under the EF1α promoter (Sigma Aldrich, St. Louis, MO, USA).

Techniques: Expressing, Variant Assay, Infection, Derivative Assay, Construct, Plasmid Preparation, Transfection, Sequencing, Western Blot, Negative Control, Control, Imaging, Positive Control

Journal: bioRxiv

Article Title: Prion infection, transmission and cytopathology modelled in a low-biohazard human cell line

doi: 10.1101/2020.04.01.019786

Figure Lengend Snippet: (A) The seeding activity of PG127-infected or NBH-treated hovS, povS and SH-SY5Y Δ PRNP lysates was assessed by RT-QuIC (diluted 1:50 and 1:250). PG127-infected hovS, and to a lesser extent povS cells, induced de novo PrP aggregate formation at both dilutions, whereas cell lysates of either NBH-treated or PG127-infected SH-SY5Y Δ PRNP used as negative controls did not yield a positive signal. CJD and non-CJD brain homogenates were used as positive and negative controls for the amplification reaction. Samples were analyzed in quadruplicates. (B) Western blot analysis of serial transmissibility. PG127-infected hovS cell lysates (PG hovS) were used to transmit prion infectivity to fresh hovS cultures. Lysates of undigested and PK-digested hovS exposed to PG127-infected hovS lysates displayed the same electrophoretic profiles as the original lysates. Lysates of hovS exposed to NBH-treated hovS and PG127-infected SH-SY5Y Δ PRNP cells were used as negative controls. NBH and PG127 inoculum were loaded as additional controls (rightmost lanes). (C) Lysates (20 µl) of PG127-infected hovS and SH-SY5Y Δ PRNP or NBH-treated hovS were intracerebrally inoculated into tg338 mice. All mice succumbed to disease upon inoculation with PG127-infected hovS lysates with an incubation time of 72 ± 2.5 days. Mice inoculated with control lysates do not show any clinical sign of disease >130 (dpi). n=6 for each condition. (D) Lysates of PG127-infected hovS (diluted 1:50) were analyzed for propagation efficiency and substrate specificity by PMCA using substrates from various species and of different genotypes (sheep VRQ/VRQ (tg338), sheep ARQ/ARQ (tgARQ), bovine (tgbov), human 129Met (tg650), human 129V (tg361)). PMCA reactions of the third round were analyzed for PrP Sc by Western blotting. Lysates of PG127-infected hovS cells showed positive seeding reactions only with the ovine substrates. NBH-treated hovS and PG127-infected SH-SY5Y ΔPRNP were used as negative controls and PG127, BSE and sCJD prions amplified with the respective substrates as positive controls. Reference: PG127 inoculum used to control for signal intensity and band shifts. One representative data set from three experiments is shown.

Article Snippet: PRNP coding sequence (CDS, 774 bp) of Ovis aries harboring the VRQ allele (GeneScript, Piscataway Townsip, NJ, USA), codon optimized for expression in human cell lines and modified to include the human ER localization signal was cloned into the expression vector (Catalog Nr: OGS606-5U) under the EF1α promoter (Sigma Aldrich, St. Louis, MO, USA).

Techniques: Activity Assay, Infection, Amplification, Western Blot, Incubation, Control

Journal: Life Science Alliance

Article Title: Prion infection, transmission, and cytopathology modeled in a low-biohazard human cell line

doi: 10.26508/lsa.202000814

Figure Lengend Snippet: (A) Generation of SH-SY5Y Δ PRNP cell lines expressing the ovine VRQ PrP C variant and its subsequent infection with the PG127 strain of sheep-derived prions passaged in tg338 mice. Hin dIII and Eco RI restriction sites were used to clone the ovine PRNP construct. Ticks in the plasmid map correspond to increments of 1,000 base pairs. hSP, human signal peptide (purple); oSP, ovine signal peptide (blue); hovS, monoclonal ovSH-SY5Y; povS, polyclonal ovSH-SY5Y; ovSH-SY5Y, SH-SY5Y Δ PRNP transfected with a plasmid harboring the sequence for ovine PRNP (ov PRNP ). (B) Western blot analysis comparing the expression levels of PrP C in hovS and povS with those in wt SH-SY5Y and in the human cell lines U251-MG and LN229. HovS and povS showed similar PrP C expression levels as U251-MG and LN229, whereas the levels in wt SH-SY5Y were slightly lower. SH-SY5Y Δ PRNP cells were used as negative control and actin as loading control. The anti-PrP antibody POM2 was used for detection. (C) Confocal imaging to detect cell surface exposed PrP C on hovS and povS. hovS and a subpopulation of povS showed a strong signal for cell surface exposed PrP C , whereas no detectable signal was visible for SH-SY5Y Δ PRNP . LN229 cells were used as positive control. The anti-PrP antibody POM1 (here and henceforth) was used for detection of PrP. Source data are available for this figure.

Article Snippet: PRNP coding sequence (CDS, 774 bp) of O. aries harboring the VRQ allele (GeneScript), codon optimized for expression in human cell lines and modified to include the human ER localization signal, was cloned into the expression vector (Cat. no. OGS606-5U) under the EF1α promoter (Sigma-Aldrich).

Techniques: Expressing, Variant Assay, Infection, Derivative Assay, Construct, Plasmid Preparation, Transfection, Sequencing, Western Blot, Negative Control, Control, Imaging, Positive Control

Journal: Life Science Alliance

Article Title: Prion infection, transmission, and cytopathology modeled in a low-biohazard human cell line

doi: 10.26508/lsa.202000814

Figure Lengend Snippet: (A) The seeding activity of PG127-infected or noninfectious brain homogenate (NBH)–treated hovS, povS, and SH-SY5Y Δ PRNP lysates was assessed by RT-QuIC (diluted 1:50 and 1:250). PG127-infected hovS, and to a lesser extent, povS cells induced de novo PrP aggregate formation at both dilutions, whereas cell lysates of either NBH-treated or PG127-infected SH-SY5Y Δ PRNP used as negative controls did not yield a positive signal. Creutzfeldt–Jakob disease (CJD) and non-CJD brain homogenates were used as positive and negative controls for the amplification reaction. Samples were analyzed in quadruplicates. (B) Western blot analysis of serial transmissibility. PG127-infected hovS cell lysates (PG hovS) were used to transmit prion infectivity to fresh hovS cultures. Lysates of undigested and proteinase K–digested hovS exposed to PG127-infected hovS lysates displayed the same electrophoretic profiles as the original lysates. Lysates of hovS exposed to NBH-treated hovS and PG127-infected SH-SY5Y Δ PRNP cells were used as negative controls. NBH and PG127 inoculum were loaded as additional controls (rightmost lanes). (C) Lysates (20 μl) of PG127-infected hovS and SH-SY5Y Δ PRNP or NBH-treated hovS were intracerebrally inoculated into tg338 mice. All mice succumbed to disease upon inoculation with PG127-infected hovS lysates with an incubation time of 72 ± 2.5 d. Mice inoculated with control lysates do not show any clinical sign of disease >130 (dpi). n = 6 for each condition. (D) Lysates of PG127-infected hovS (diluted 1:50) were analyzed for propagation efficiency and substrate specificity by PMCA using substrates from various species and of different genotypes (sheep VRQ/VRQ [tg338], sheep ARQ/ARQ [tgARQ], bovine [tgbov], human 129Met [tg650], and human 129V [tg361]). PMCA reactions of the third round were analyzed for PrP Sc by Western blotting. Lysates of PG127-infected hovS cells showed positive seeding reactions only with the ovine substrates. NBH-treated hovS and PG127-infected SH-SY5Y ΔPRNP were used as negative controls and PG127, BSE, and sCJD prions amplified with the respective substrates as positive controls. Reference: PG127 inoculum used to control for signal intensity and band shifts. One representative data set from three experiments is shown. Source data are available for this figure.

Article Snippet: PRNP coding sequence (CDS, 774 bp) of O. aries harboring the VRQ allele (GeneScript), codon optimized for expression in human cell lines and modified to include the human ER localization signal, was cloned into the expression vector (Cat. no. OGS606-5U) under the EF1α promoter (Sigma-Aldrich).

Techniques: Activity Assay, Infection, Amplification, Western Blot, Incubation, Control