cd55 cdna  (OriGene)

Journal: Frontiers in Immunology

Article Title: The Sez6 Family Inhibits Complement by Facilitating Factor I Cleavage of C3b and Accelerating the Decay of C3 Convertases

doi: 10.3389/fimmu.2021.607641

Figure Lengend Snippet: Sez6 family expression in the hippocampus. (A) Sez6, Sez6L, and Sez6L2 are expressed by principal (excitatory, pyramidal) neurons of the mouse hippocampus at much higher levels than other known complement regulators (namely Crry, C4BP, CFH, C1-INH, DAF, and MCP). Expression data was obtained from Hipposeq: a comprehensive RNA-Seq database of gene expression in hippocampal principal neurons [ http://hipposeq.janelia.org ]. The RNA samples used in this database were isolated from mouse hippocampal principal neurons micro-dissected from the CA1, CA3, or Dentate Gyrus (DG) cell layers of the hippocampus at Postnatal Day 25-32. Differential gene expression is shown in the heatmap with the relative units of FPKM (Fragments per Kilobase of Exon per Million Reads Mapped.) (B) Brain sections from adult WT mice or Sez6 triple knockout mice (TKO) were immuno-stained for Sez6L2 (green) and DAPI and imaged in the CA1 region of the hippocampus. Scale Bar= 27 µm. High density Sez6L2 staining occurs around cell bodies in the pyramidal layer, but significant Sez6L2 is also found in the stratum radiatum and stratum oriens. (C) Higher magnification images of sections immuno-stained for Sez6L2 (green) and the postsynaptic protein, Homer1 (red), shows a subset of Sez6L2 is found near or co-localized with synapses in the stratum radiatum. Scale bar = 1.8 µm.

Article Snippet: Alternatively, cells were transfected with a GFP expression plasmid or co-transfected with GFP and M-SEZ6L2 or HIS-tagged DAF (H-DAF; Sino Biological HG10101-NH; modified from NCBI RefSeq NM_000574.3).

Techniques: Expressing, RNA Sequencing Assay, Isolation, Triple Knockout, Staining

Journal: Frontiers in Immunology

Article Title: The Sez6 Family Inhibits Complement by Facilitating Factor I Cleavage of C3b and Accelerating the Decay of C3 Convertases

doi: 10.3389/fimmu.2021.607641

Figure Lengend Snippet: Full Length Sez6L2, Sez6, and Sez6L inhibit C3b/iC3b opsonization of CHO cells by the classical pathway. (A, B) Sez6L2 inhibits C3b/iC3b opsonization at a range of serum concentrations. CHO cells were transfected with plasmids for GFP alone or with Myc-tagged Sez6L2 (M-Sez6L2) or His-tagged DAF (H-DAF). CHO cells were coated with antibodies and exposed to 0-20% C5-depleted human serum for one hour and then immuno-stained with anti-C3b/iC3b antibodies and analyzed by flow cytometry. One experiment is shown that is representative of two independent experiments. (B) C3b/iC3b on GFP transfected cells with or without M-Sez6L2 or H-DAF at 15% serum. ANOVA (P=0.0016; F(2,6)=22.51). N=3; one experiment with three replicates (representative of 3+ independent experiments). (C) Schematic of Sez6L2, Sez6, and Sez6L protein domain structures. (D–I) CHO cells were transfected with the indicated Myc-tagged cDNAs and processed as outlined in A with 15% C5 depleted serum, except that an anti-Myc antibody was used in place of GFP to identify transfected and expressing CHO cells. (D) 5% Contour plots of C3b/iC3b versus Myc fluorescence (top layer) and C3b/iC3b fluorescence histograms (bottom layer) of the same samples normalized to mode and compared to baseline cells not exposed to serum. For Contour plots, boxed regions highlight cells designated as Myc-positive (top box) and Myc-negative (lower box) populations. For C3b/iC3b histograms, dark grey, solid line population = Myc-positive cells; Light grey, dotted line population= Myc-negative cells; White, dashed grey line population = baseline. Representative of 4+ independent experiments. (E) Quantification of the average median C3b/iC3b fluorescence intensity from Myc-positive and Myc-negative cells within each sample. Statistics = t-tests. N=3 (one experiment with three replicates; Representative of 4+ independent experiments). (F) Average median C3b/iC3b fluorescence intensities after normalization to the Myc-negative cells from each experimental group. ANOVA between Myc-positive cell populations (p<0.001; F(4, 15)=64.53). Sez6L2 inhibits C3b/iC3b opsonization at a level comparable to positive control MCP. Sez6 is a stronger complement inhibitor than Sez6L2 and Sez6L is a weaker inhibitor. (F) Average median Myc fluorescence intensity from Myc-positive cells. ANOVA (p<0.001; F(4, 15)=36.79). (G) Average % of Myc-positive cells in each experimental group (ANOVA, p=0.115; F(4, 15)=2.224). For sections (F–H) , N=4 (four independent experiments). (I) Sez6 blocks complement opsonization more efficiently than Sez6L2 and Sez6L even when comparing similar levels of Myc surface expression. Average C3b/iC3b median fluorescence intensity normalized to internal Myc-negative populations for M-Sez6, M-Sez6L2, and M-Sez6L samples shown relative to the Myc median fluorescence intensity. N=3 (one experiment with three replicates, Representative of three independent experiments). For all graphs *p < 0.05; **p < 0.01; # p < 0.001 for all Myc-positive groups compared to M-CR2.

Article Snippet: Alternatively, cells were transfected with a GFP expression plasmid or co-transfected with GFP and M-SEZ6L2 or HIS-tagged DAF (H-DAF; Sino Biological HG10101-NH; modified from NCBI RefSeq NM_000574.3).

Techniques: Transfection, Staining, Flow Cytometry, Expressing, Fluorescence, Positive Control

Journal: Frontiers in Immunology

Article Title: The Sez6 Family Inhibits Complement by Facilitating Factor I Cleavage of C3b and Accelerating the Decay of C3 Convertases

doi: 10.3389/fimmu.2021.607641

Figure Lengend Snippet: Truncated Sez6L2 inhibits alternative pathway hemolysis more than classical pathway hemolysis. (A) Schematic of Sez6L2 and Sez6L2-MH domain structures. CCP=Domain abundant in complement control proteins. CCP domains are also known as SUSHI repeats or short complement-like repeat (SCR). CUB= Domains named after complement C1r/C1s, uEGF, and BMP1. TM=Transmembrane region. Sez6L2-MH was made by replacing the transmembrane and cytoplasmic tail domains with a tandem Myc, 6xHis tag. (B) Purified Sez6L2-MH is shown by a Coomassie stained gel and by western blot with anti-Sez6L2 and anti-Myc antibodies. Lanes with the Coomassie stain are from the same gel. (C) Classical pathway hemolysis assay. Antibody-coated sheep erythrocytes were exposed to human serum pre-incubated with purified Sez6L2-MH, H-DAF, FH, C4BP, C1-INH, or BSA. After 30 mins, the percent of cell lysis was measured by spectrophotometry (A415). One-way ANOVA (P <0.0001; F(6,30)=233.2); PBS N=10; Sez6L2-MH N=7; H-DAF N=4; CFH N=4; C4BP N=3; C1-INH N=3; BSA N=6. (D) Alternative pathway hemolysis assay. Rabbit erythrocytes were exposed to human serum pre-incubated with Sez6L2-MH, complement regulators, or BSA in presence of 10 mM MgEGTA to block the classical pathway. Then the percent of cell lysis was measured by spectrophotometry (A415). One-way ANOVA (P <0.0001; F(4,16)=33.88). PBS N=6, Sez6L2-MH N=5; FH N=2, H-DAF N=2, and BSA N=6. For all graphs *p < 0.05 compared to PBS control and # p < 0.05 compared to BSA negative control.

Article Snippet: Alternatively, cells were transfected with a GFP expression plasmid or co-transfected with GFP and M-SEZ6L2 or HIS-tagged DAF (H-DAF; Sino Biological HG10101-NH; modified from NCBI RefSeq NM_000574.3).

Techniques: Purification, Staining, Western Blot, Hemolysis Assay, Incubation, Lysis, Spectrophotometry, Blocking Assay, Negative Control

Journal: Frontiers in Immunology

Article Title: The Sez6 Family Inhibits Complement by Facilitating Factor I Cleavage of C3b and Accelerating the Decay of C3 Convertases

doi: 10.3389/fimmu.2021.607641

Figure Lengend Snippet: Truncated Sez6L2 has decay accelerating activity for the alternative pathway C3 convertase but has only modest decay accelerating activity for the classical/lectin pathway C3 convertase. (A–D) Alternative C3 convertase assay: (A–C) A 96 well plate coated with C3b was incubated with Factor B and Factor D to form the C3 convertase C3bBb, then incubated with Sez6L2-MH or FH at various concentrations ranging from 0-6000 nM (equivalent to 0-500 µg/mL) to assess their decay accelerating activity. Factor B remaining bound to the plate (as C3bBb) was detected using an anti-Factor B antibody ELISA in (A) and Bb released into the supernatant is shown via western blot in (B) with Bb band densities quantified in (C) . For the western blot and quantification, Sez6L2-MH and FH concentrations are listed in both nM and µg/mL. Quantification of Bb band densities were normalized to the 0 nM control lanes. N=3 samples per group. (D) The alternative C3 convertase decay ELISA described above was repeated comparing 0, 1uM, and 5uM of Sez6L2-MH, H-DAF, FH, and BSA. (E) Classical C3 convertase assay: A plate coated with C4b was incubated with C2 and C1s-enzyme to form the classical/lectin pathway C3 convertase C4b2b, then incubated with Sez6L2-MH, H-DAF at concentrations ranging from 0 to 7000 nM to assess their decay accelerating activity. C2 remaining bound to the plate (presumably as C4b2b) was detected using an anti-C2 antibody ELISA. For A, D, and E ELISAs: N=3 (1 experiment with 3 replicates; representative of 2-3 independent experiments). Statistics: one-way ANOVAS with Holms-Sidak multiple comparison’s tests to controls. *p < 0.05, **< 0.01.

Article Snippet: Alternatively, cells were transfected with a GFP expression plasmid or co-transfected with GFP and M-SEZ6L2 or HIS-tagged DAF (H-DAF; Sino Biological HG10101-NH; modified from NCBI RefSeq NM_000574.3).

Techniques: Activity Assay, Convertase Assay, Incubation, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: The Sez6 Family Inhibits Complement by Facilitating Factor I Cleavage of C3b and Accelerating the Decay of C3 Convertases

doi: 10.3389/fimmu.2021.607641

Figure Lengend Snippet: Sez6 family expression in the hippocampus. (A) Sez6, Sez6L, and Sez6L2 are expressed by principal (excitatory, pyramidal) neurons of the mouse hippocampus at much higher levels than other known complement regulators (namely Crry, C4BP, CFH, C1-INH, DAF, and MCP). Expression data was obtained from Hipposeq: a comprehensive RNA-Seq database of gene expression in hippocampal principal neurons [ http://hipposeq.janelia.org ]. The RNA samples used in this database were isolated from mouse hippocampal principal neurons micro-dissected from the CA1, CA3, or Dentate Gyrus (DG) cell layers of the hippocampus at Postnatal Day 25-32. Differential gene expression is shown in the heatmap with the relative units of FPKM (Fragments per Kilobase of Exon per Million Reads Mapped.) (B) Brain sections from adult WT mice or Sez6 triple knockout mice (TKO) were immuno-stained for Sez6L2 (green) and DAPI and imaged in the CA1 region of the hippocampus. Scale Bar= 27 µm. High density Sez6L2 staining occurs around cell bodies in the pyramidal layer, but significant Sez6L2 is also found in the stratum radiatum and stratum oriens. (C) Higher magnification images of sections immuno-stained for Sez6L2 (green) and the postsynaptic protein, Homer1 (red), shows a subset of Sez6L2 is found near or co-localized with synapses in the stratum radiatum. Scale bar = 1.8 µm.

Article Snippet: Alternatively, cells were transfected with a GFP expression plasmid or co-transfected with GFP and M-SEZ6L2 or HIS-tagged DAF (H-DAF; Sino Biological HG10101-NH; modified from NCBI RefSeq NM_000574.3).

Techniques: Expressing, RNA Sequencing Assay, Isolation, Triple Knockout, Staining

Journal: Frontiers in Immunology

Article Title: The Sez6 Family Inhibits Complement by Facilitating Factor I Cleavage of C3b and Accelerating the Decay of C3 Convertases

doi: 10.3389/fimmu.2021.607641

Figure Lengend Snippet: Full Length Sez6L2, Sez6, and Sez6L inhibit C3b/iC3b opsonization of CHO cells by the classical pathway. (A, B) Sez6L2 inhibits C3b/iC3b opsonization at a range of serum concentrations. CHO cells were transfected with plasmids for GFP alone or with Myc-tagged Sez6L2 (M-Sez6L2) or His-tagged DAF (H-DAF). CHO cells were coated with antibodies and exposed to 0-20% C5-depleted human serum for one hour and then immuno-stained with anti-C3b/iC3b antibodies and analyzed by flow cytometry. One experiment is shown that is representative of two independent experiments. (B) C3b/iC3b on GFP transfected cells with or without M-Sez6L2 or H-DAF at 15% serum. ANOVA (P=0.0016; F(2,6)=22.51). N=3; one experiment with three replicates (representative of 3+ independent experiments). (C) Schematic of Sez6L2, Sez6, and Sez6L protein domain structures. (D–I) CHO cells were transfected with the indicated Myc-tagged cDNAs and processed as outlined in A with 15% C5 depleted serum, except that an anti-Myc antibody was used in place of GFP to identify transfected and expressing CHO cells. (D) 5% Contour plots of C3b/iC3b versus Myc fluorescence (top layer) and C3b/iC3b fluorescence histograms (bottom layer) of the same samples normalized to mode and compared to baseline cells not exposed to serum. For Contour plots, boxed regions highlight cells designated as Myc-positive (top box) and Myc-negative (lower box) populations. For C3b/iC3b histograms, dark grey, solid line population = Myc-positive cells; Light grey, dotted line population= Myc-negative cells; White, dashed grey line population = baseline. Representative of 4+ independent experiments. (E) Quantification of the average median C3b/iC3b fluorescence intensity from Myc-positive and Myc-negative cells within each sample. Statistics = t-tests. N=3 (one experiment with three replicates; Representative of 4+ independent experiments). (F) Average median C3b/iC3b fluorescence intensities after normalization to the Myc-negative cells from each experimental group. ANOVA between Myc-positive cell populations (p<0.001; F(4, 15)=64.53). Sez6L2 inhibits C3b/iC3b opsonization at a level comparable to positive control MCP. Sez6 is a stronger complement inhibitor than Sez6L2 and Sez6L is a weaker inhibitor. (F) Average median Myc fluorescence intensity from Myc-positive cells. ANOVA (p<0.001; F(4, 15)=36.79). (G) Average % of Myc-positive cells in each experimental group (ANOVA, p=0.115; F(4, 15)=2.224). For sections (F–H) , N=4 (four independent experiments). (I) Sez6 blocks complement opsonization more efficiently than Sez6L2 and Sez6L even when comparing similar levels of Myc surface expression. Average C3b/iC3b median fluorescence intensity normalized to internal Myc-negative populations for M-Sez6, M-Sez6L2, and M-Sez6L samples shown relative to the Myc median fluorescence intensity. N=3 (one experiment with three replicates, Representative of three independent experiments). For all graphs *p < 0.05; **p < 0.01; # p < 0.001 for all Myc-positive groups compared to M-CR2.

Article Snippet: Alternatively, cells were transfected with a GFP expression plasmid or co-transfected with GFP and M-SEZ6L2 or HIS-tagged DAF (H-DAF; Sino Biological HG10101-NH; modified from NCBI RefSeq NM_000574.3).

Techniques: Transfection, Staining, Flow Cytometry, Expressing, Fluorescence, Positive Control

Journal: Frontiers in Immunology

Article Title: The Sez6 Family Inhibits Complement by Facilitating Factor I Cleavage of C3b and Accelerating the Decay of C3 Convertases

doi: 10.3389/fimmu.2021.607641

Figure Lengend Snippet: Full Length Sez6L2, Sez6, and Sez6L inhibit C3b/iC3b opsonization of CHO cells by the alternative pathway. CHO cells were transfected with the indicated Myc-tagged cDNAs and then coated with a low level of antibodies and exposed to 20% C5-depleted human serum for one hour in the presence of 10 mM EGTA and 10 mM MgCl 2 to block the classical pathway. Cells were then labeled with anti-C3b/iC3b and anti-Myc antibodies and analyzed by flow cytometry. (A) 5% Contour plots of C3b/iC3b versus Myc fluorescence (top layer) and C3b/iC3b fluorescence histograms (bottom layer) of the same samples normalized to mode and compared to baseline cells not exposed to serum. For Contour plots, boxed regions highlight cells designated as Myc-positive (top box) and Myc-negative (lower box) populations. For C3b/iC3b histograms, dark grey, solid line population = Myc-positive cells; Light grey, dotted line population= Myc-negative cells; White, dashed grey line population = baseline. Representative of 3+ independent experiments with technical replicates. (B) Quantification of the average median C3b/iC3b fluorescence intensity from Myc-positive and Myc-negative cells within each sample. N=3 (one experiment with three replicates; Representative of 3+ independent experiments) Statistics = t-tests. E) Average median C3b/iC3b fluorescence intensities after normalization to the Myc-negative cells from each experimental group. ANOVA (p<0.001; F(4, 10)=74.47. N=3 (3 independent experiments). For all graphs *p < 0.05; **p < 0.01 # p < 0.001 for all Myc-positive groups compared to M-CR2.

Article Snippet: Alternatively, cells were transfected with a GFP expression plasmid or co-transfected with GFP and M-SEZ6L2 or HIS-tagged DAF (H-DAF; Sino Biological HG10101-NH; modified from NCBI RefSeq NM_000574.3).

Techniques: Transfection, Blocking Assay, Labeling, Flow Cytometry, Fluorescence

Journal: Frontiers in Immunology

Article Title: The Sez6 Family Inhibits Complement by Facilitating Factor I Cleavage of C3b and Accelerating the Decay of C3 Convertases

doi: 10.3389/fimmu.2021.607641

Figure Lengend Snippet: Truncated Sez6L2 inhibits alternative pathway hemolysis more than classical pathway hemolysis. (A) Schematic of Sez6L2 and Sez6L2-MH domain structures. CCP=Domain abundant in complement control proteins. CCP domains are also known as SUSHI repeats or short complement-like repeat (SCR). CUB= Domains named after complement C1r/C1s, uEGF, and BMP1. TM=Transmembrane region. Sez6L2-MH was made by replacing the transmembrane and cytoplasmic tail domains with a tandem Myc, 6xHis tag. (B) Purified Sez6L2-MH is shown by a Coomassie stained gel and by western blot with anti-Sez6L2 and anti-Myc antibodies. Lanes with the Coomassie stain are from the same gel. (C) Classical pathway hemolysis assay. Antibody-coated sheep erythrocytes were exposed to human serum pre-incubated with purified Sez6L2-MH, H-DAF, FH, C4BP, C1-INH, or BSA. After 30 mins, the percent of cell lysis was measured by spectrophotometry (A415). One-way ANOVA (P <0.0001; F(6,30)=233.2); PBS N=10; Sez6L2-MH N=7; H-DAF N=4; CFH N=4; C4BP N=3; C1-INH N=3; BSA N=6. (D) Alternative pathway hemolysis assay. Rabbit erythrocytes were exposed to human serum pre-incubated with Sez6L2-MH, complement regulators, or BSA in presence of 10 mM MgEGTA to block the classical pathway. Then the percent of cell lysis was measured by spectrophotometry (A415). One-way ANOVA (P <0.0001; F(4,16)=33.88). PBS N=6, Sez6L2-MH N=5; FH N=2, H-DAF N=2, and BSA N=6. For all graphs *p < 0.05 compared to PBS control and # p < 0.05 compared to BSA negative control.

Article Snippet: Alternatively, cells were transfected with a GFP expression plasmid or co-transfected with GFP and M-SEZ6L2 or HIS-tagged DAF (H-DAF; Sino Biological HG10101-NH; modified from NCBI RefSeq NM_000574.3).

Techniques: Purification, Staining, Western Blot, Hemolysis Assay, Incubation, Lysis, Spectrophotometry, Blocking Assay, Negative Control

Journal: Frontiers in Immunology

Article Title: The Sez6 Family Inhibits Complement by Facilitating Factor I Cleavage of C3b and Accelerating the Decay of C3 Convertases

doi: 10.3389/fimmu.2021.607641

Figure Lengend Snippet: Truncated Sez6L2 is a cofactor for Factor I cleavage of C3b. (A ) Schematic of Factor I and cofactor cleavage of C3b and iC3b. (B) C3b and Factor I (FI) were incubated alone, with concentrations of Sez6L2-MH ranging from 1 to 8 µM, or with 1 µM Factor H (FH) or C4BP for two hours at 37°C. Then samples were analyzed by western blot using antibodies that recognize C3d, a region within the C3α chain [and highlighted by the black rectangle in the schematics in (A) ]. Coomassie stained gels are also shown. Sez6L2-MH supports partial FI cleavage of C3b at sites 1 and 2 generating the cleavage products C3α’1 and C3α’2 in a concentration dependent manner. FH and C4BP (known co-factors of FI towards C3b) supported almost full cleavage of C3b at sites 1 and 2. FH also showed partial cleavage at site 3 generating some C3dg and C3c-C3α’1. (C) C3b and FI were incubated with 1.5 µM Sez6L2-MH for 0.25-8 hours and then were visualized by western blot and Coomassie stained gels. Sez6L2-MH facilitates more FI cleavage with increased time including some partial cleavage at site 3 to generating C3dg and C3c-C3α’1.

Article Snippet: Alternatively, cells were transfected with a GFP expression plasmid or co-transfected with GFP and M-SEZ6L2 or HIS-tagged DAF (H-DAF; Sino Biological HG10101-NH; modified from NCBI RefSeq NM_000574.3).

Techniques: Incubation, Western Blot, Staining, Concentration Assay

Journal: Frontiers in Immunology

Article Title: The Sez6 Family Inhibits Complement by Facilitating Factor I Cleavage of C3b and Accelerating the Decay of C3 Convertases

doi: 10.3389/fimmu.2021.607641

Figure Lengend Snippet: Truncated Sez6L2 does not act as a cofactor for Factor I cleavage of C4b. (A) Schematic of Factor I plus cofactor cleavage of C4b. (B) C4b and FI were incubated alone, with concentrations of Sez6L2-MH ranging from 1 to 8 µM, or with 1 µM C4BP for two hours at 37°C. Then samples were then run and visualized on Coomassie stained gels. Incubation of C4b and FI with Sez6L2-MH did not result in the appearance of C4b cleavage products C4d, C4α3, or C4α4. On the other hand, C4BP (a known cofactor of FI for C4b cleavage) supported FI’s production of C4d, C4α3, and C4α4. (C) Sez6L2-MH does not support Factor I cleavage of C4b even with increased time. C4b and FI were incubated with 1.5 µM Sez6L2-MH for 0.25-8 hours and then visualized on Coomassie stained gels.

Article Snippet: Alternatively, cells were transfected with a GFP expression plasmid or co-transfected with GFP and M-SEZ6L2 or HIS-tagged DAF (H-DAF; Sino Biological HG10101-NH; modified from NCBI RefSeq NM_000574.3).

Techniques: Incubation, Staining

Journal: Frontiers in Immunology

Article Title: The Sez6 Family Inhibits Complement by Facilitating Factor I Cleavage of C3b and Accelerating the Decay of C3 Convertases

doi: 10.3389/fimmu.2021.607641

Figure Lengend Snippet: Truncated Sez6L2 has decay accelerating activity for the alternative pathway C3 convertase but has only modest decay accelerating activity for the classical/lectin pathway C3 convertase. (A–D) Alternative C3 convertase assay: (A–C) A 96 well plate coated with C3b was incubated with Factor B and Factor D to form the C3 convertase C3bBb, then incubated with Sez6L2-MH or FH at various concentrations ranging from 0-6000 nM (equivalent to 0-500 µg/mL) to assess their decay accelerating activity. Factor B remaining bound to the plate (as C3bBb) was detected using an anti-Factor B antibody ELISA in (A) and Bb released into the supernatant is shown via western blot in (B) with Bb band densities quantified in (C) . For the western blot and quantification, Sez6L2-MH and FH concentrations are listed in both nM and µg/mL. Quantification of Bb band densities were normalized to the 0 nM control lanes. N=3 samples per group. (D) The alternative C3 convertase decay ELISA described above was repeated comparing 0, 1uM, and 5uM of Sez6L2-MH, H-DAF, FH, and BSA. (E) Classical C3 convertase assay: A plate coated with C4b was incubated with C2 and C1s-enzyme to form the classical/lectin pathway C3 convertase C4b2b, then incubated with Sez6L2-MH, H-DAF at concentrations ranging from 0 to 7000 nM to assess their decay accelerating activity. C2 remaining bound to the plate (presumably as C4b2b) was detected using an anti-C2 antibody ELISA. For A, D, and E ELISAs: N=3 (1 experiment with 3 replicates; representative of 2-3 independent experiments). Statistics: one-way ANOVAS with Holms-Sidak multiple comparison’s tests to controls. *p < 0.05, **< 0.01.

Article Snippet: Alternatively, cells were transfected with a GFP expression plasmid or co-transfected with GFP and M-SEZ6L2 or HIS-tagged DAF (H-DAF; Sino Biological HG10101-NH; modified from NCBI RefSeq NM_000574.3).

Techniques: Activity Assay, Convertase Assay, Incubation, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Stem cell research

Article Title: Conditional gene knockout and reconstitution in human iPSCs with an inducible Cas9 system.

doi: 10.1016/j.scr.2018.03.003

Figure Lengend Snippet: Fig. 2. Efficient knockout of CD55 in human iPSC line BC1-AiCas9. A: Schematic of CRISPR-mediated gene targeting in exon 1 of the CD55 gene. The location of the ATG start codon is also indicated. Two Sequences recognized by guide RNAs (gRNA) are shown. PAM sequences are in green; orange arrowheads indicate the cut sites. B: A timeline of approach by adding doxycycline (+dox) for induction and gRNA delivery. C: Flow cytometric analysis of CD55 four days after gRNA electroporation. The cells lacking CD55 expression (in red box) is likely due to the knock-out (KO) in both alleles. D: A gel image of CD55 PCR amplicons prepared from individually picked 24 colonies, showing that at least three clones (#9, 21 and 23) that only have KO alleles with deletion. E: Flow cytometric analysis of the three selected clones that have deletions in both alleles (homozygous) as the CD55 KO clones. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The coding sequence (CDS) of CD55 was PCR-amplified from the template pCMV6-AC-CD55 containing CD55 cDNA (Origene, #SC322226).We next inserted CD55 CDS into the PB-TRE piggyBac vector (Addgene plasmid #63800) which contains a tet-on inducible promoter (TRE3G) and a hygromycin selection cassette (Randolph et al., 2017), to generate the PB-TRE-CD55 plasmid.

Techniques: Knock-Out, CRISPR, Electroporation, Expressing, Clone Assay

Journal: Stem cell research

Article Title: Conditional gene knockout and reconstitution in human iPSCs with an inducible Cas9 system.

doi: 10.1016/j.scr.2018.03.003

Figure Lengend Snippet: Fig. 3. Selected CD55 KO iPSCs are phenotypically normal and pluripotent. A: Representative images of undifferentiated phenotypes of the CD55KO iPSC line, clone 21. It expressed pluripotency markers: TRA-1-60, OCT4 and NANOG, nucleus was labelled with DAPI. B: Karyotyping of CD55KO AiCas9. C: Pluripotency test in vitro by embryoid body formation followed by differentiation. Immunostaining analysis after differentiation showed the cells derived all three embryonic germ layers endoderm, mesoderm and ectoderm, demonstrating the pluripotency of CD55KO AiCas9. Scale bar: 100 μm.

Article Snippet: The coding sequence (CDS) of CD55 was PCR-amplified from the template pCMV6-AC-CD55 containing CD55 cDNA (Origene, #SC322226).We next inserted CD55 CDS into the PB-TRE piggyBac vector (Addgene plasmid #63800) which contains a tet-on inducible promoter (TRE3G) and a hygromycin selection cassette (Randolph et al., 2017), to generate the PB-TRE-CD55 plasmid.

Techniques: In Vitro, Immunostaining, Derivative Assay

Journal: Stem cell research

Article Title: Conditional gene knockout and reconstitution in human iPSCs with an inducible Cas9 system.

doi: 10.1016/j.scr.2018.03.003

Figure Lengend Snippet: Fig. 4. Reconstitute conditional CD55 expression by using a plasmid that contains a tet-on promoter (TRE-3G) and the piggyBac transposon. A: The presence of the M2rtTA activator in the AiCas9 KO iPSCs, which is required for the Tet-On mediated expression of a CD55 transgene. B: Flow cytometry analysis of the cells after lentiviral transduction and doxycycline (dox) induction. The enriched CD55+ cells after cell sorting is also shown. C: The kinetics of CD55 expression on cell surface over time with or without dox inducer. D: Cells expressed pluripotency markers: TRA-1-60, OCT4 and NANOG, nucleus was labelled with DAPI. E: Pluripotency test in vitro by embryoid body formation. Scale bar: 100 μm. F: A normal karyotype (46, XY) of representative reconstituted AiCas9 iPSC clone, after CD55 KO and the piggyBac-mediated reconstitution, at passage 42 of cell expansion.

Article Snippet: The coding sequence (CDS) of CD55 was PCR-amplified from the template pCMV6-AC-CD55 containing CD55 cDNA (Origene, #SC322226).We next inserted CD55 CDS into the PB-TRE piggyBac vector (Addgene plasmid #63800) which contains a tet-on inducible promoter (TRE3G) and a hygromycin selection cassette (Randolph et al., 2017), to generate the PB-TRE-CD55 plasmid.

Techniques: Expressing, Plasmid Preparation, Flow Cytometry, Transduction, FACS, In Vitro

Journal: Stem cell research

Article Title: Conditional gene knockout and reconstitution in human iPSCs with an inducible Cas9 system.

doi: 10.1016/j.scr.2018.03.003

Figure Lengend Snippet: Fig. 5. Characterization of inducible CD55 expression in engineered human iPSCs after extended culture. A: Flow cytometry analysis of inducible CD55 expression from the piggyBac vector in transgenic iPSCs after one month culture. B: A differentiation strategy from iPSCs by embryoid body (EB) formation to generate hematopoietic progenitor cells (HPCs). HPCs present in the suspension of day 14 culture were harvested and induced by Doxycycline (Dox). C: Flow cytometry analysis of CD55 in iPSC-derived CD34 + CD45+ HPCs with or without Dox induction for 2 days.

Article Snippet: The coding sequence (CDS) of CD55 was PCR-amplified from the template pCMV6-AC-CD55 containing CD55 cDNA (Origene, #SC322226).We next inserted CD55 CDS into the PB-TRE piggyBac vector (Addgene plasmid #63800) which contains a tet-on inducible promoter (TRE3G) and a hygromycin selection cassette (Randolph et al., 2017), to generate the PB-TRE-CD55 plasmid.

Techniques: Expressing, Flow Cytometry, Plasmid Preparation, Transgenic Assay, Suspension, Derivative Assay