Journal: Cell

Article Title: Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

doi: 10.1016/j.cell.2019.08.013

Figure Lengend Snippet: A) Standard postnatal EP protocol targeting the ventricular zone in P2 heterozygous Rosa26WT/mTmG pups with DNA mixture of a Flp-Cre vector and a donor plasmid B) Postnatal EP recapitulates in vitro nucleofection experiment and yields TagBFP2+ MADR along with EGFP+ and tdTomato+ lineages at 2 weeks post-EP. Scale bar, 100μm C) Different concentrations of recombinase and donor plasmids result in various efficiencies of both MADR and Cre-excision recombination reactions in vivo. All mixtures contained a nuclear TagBfp2 reporter plasmid. (See Supp. Fig. 2D for representative images from this quantitation). Error bars indicate standard error of the mean (SEM). D) Schematic of plasmid delivery for combinatorial MADR MAX “brainbow” like multiplex labeling E) Low mag image of olfactory bulb displaying multiplex smFP-based MADR MAX EPed cells and immunostaining for the smFP-linked epitope tags. Scale bar, 100μm F) High mag image of cells from E exhibiting expression of a single smFP epitope tag per neuron. Scale bar, 10μm G) Schematic of expansion microscopy and brightfield image example H) MADR pDonor smFP-myc sh.Nf1 miR-E plasmid for simultaneous knockdown of Nf1 and smFP-myc labeling of transgenic cells I) Image of EPed striatum showing two populations of reporter labeled cells—EGFP and smFP-myc (i.e. Nf1 knockdown cells). J) Pre-expansion smFP-myc cell body K) Post-expansion of cell in J L) Post-expansion EGFP astrocyte displaying “super-resolution” detail. M) Schematic of MADR pDonor-TagBFP2-P2A-VCre and FlEx VCre reporter plasmids for mosaic analysis with tertiary recombinase N) EPed striatum with FlpO-2A-Cre, MADR pDonor-TagBFP2, HypBase and FlEx VCre reporter. Scale bar, 50μm O) Striatum of littermate of mouse shown in N with FlpO-2A-Cre, MADR pDonor-TagBFP2–2A-VCre, HypBase and FlEx VCre reporter exhibiting VCre-dependent FlEx reporter (smFP-myc). Scale bar, 50μm

Article Snippet: plasmid: MADR pDonor-smFP-Myc (dark) WPRE , This paper , Addgene Plasmid #130987.

Techniques: Plasmid Preparation, In Vitro, In Vivo, Quantitation Assay, Multiplex Assay, Labeling, Immunostaining, Expressing, Microscopy, Knockdown, Transgenic Assay

Journal: Cell

Article Title: Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

doi: 10.1016/j.cell.2019.08.013

Figure Lengend Snippet: A) Donor construct for miR-E shRNAs against Nf1, Pten, and Trp53 tied to TagBFP2 reporter B) Validation of knockdown efficacy of multi-miR-E function by qPCR. C) 6-month-old mouse sagittal section showing a hyperplasia of TagBFP2+ cells but no tumor. Scale bar, 1mm D) Plasmid for MADR of a TagBFP2-V5 reporter protein and SpCas9 E) Sequencing of TdTomato-/EGFP- glioma cells exhibit InDels in Nf1 and Trp53. F) MADR insertion of TagBFP2-V5 reporter and Cas9 with co-EPed PCR-derived sgRNAs yields high grade glioma observable through labeling of 3 genetic reporter-defined populations in a coronal section of both hemispheres. Scale bar, 1000μm G) Plasmid for MADR of an smFP-myc reporter protein and FNLS Cas9n base editor. H) sgRNA-targeting sites (green letters) induce C->T base conversion (red lowercase ‘c’ are targeted) to produce premature stop codons in Nf1, Trp53, and Pten. I) MADR insertion of myc reporter and FNLS Cas9n with co-EPed PCR-derived sgRNAs yields observable expansion of OPC progenitors at two months post-EP through labeling of three genetic reporter-defined populations in a coronal section. Scale bar, 1000μm J) High magnification tdTomato (1), EGFP (2), and Myc tag (3) image showing myc+ populations. Scale bar, 100μm

Article Snippet: plasmid: MADR pDonor-smFP-Myc (dark) WPRE , This paper , Addgene Plasmid #130987.

Techniques: Construct, Biomarker Discovery, Knockdown, Plasmid Preparation, Sequencing, Derivative Assay, Labeling

Journal: Cell

Article Title: Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

doi: 10.1016/j.cell.2019.08.013

Figure Lengend Snippet: A-B) Schematic for in utero EP of MADR into E14.5 RCE +/− dams C) In utero EP in RCE mice with HrasG12V oncogene produces mosaic patches of TagBFP+ astrocytes Rosa26HrasG12 but not evidence of invasive glioma D) Schematic of possible outcomes after MADR in homozygous mt/mg recipient mice E) P2 EP of homozygous mt/mg mice with TagBFP2-HrasG12V oncogene F) Postnatal EP in homozygous Rosa26mTmG P2 pups with HrasG12V oncogene produces two different tumor types (Blue-only Rosa26HrasG12V×2 and blue-and-green Rosa26HrasG12V×1) Scale bars: 2mm G) Representative tumor formation in homozygous mTmG 3 months post-EP. Blue-only Rosa26HrasG12V×2 cells occupy a larger section of the tumor than blue-and-green Rosa26HrasG12V×1, correlating with phosphor-Rb1 protein expression. Scale bars: 1mm H) Zoom-in images of regions 1 and 2 from G show phosphorylated-Rb1 expression correlates largely with blue-only cells. Scale bars: 50μm I) Plasmid schematics for expression of ependymoma-associated fusion proteins J) Stitch of YAP1-MAML1D; p16/p19 Cas9 targeting induced ependymoma-like tumor. K) Survival analysis of Ependymoma MADR model mice L) Ependymoma-like tumor in a 3-month-old C11orf95-RELA; p16/p19 Cas9-targeted mouse

Article Snippet: plasmid: MADR pDonor-smFP-Myc (dark) WPRE , This paper , Addgene Plasmid #130987.

Techniques: In Utero, Expressing, Plasmid Preparation

Journal: Cell

Article Title: Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

doi: 10.1016/j.cell.2019.08.013

Figure Lengend Snippet: A) Schematic of donor plasmid for MADR with multiple recurrent pediatric glioma driver mutations B) Schematic of the plasmid delivery and electrode sweep employed to target striatal and cortical germinal niches simultaneously C) Zoomed view from B showing the respective cortical (magenta) and striatal (orange) germinal niches that are targeted D) Representative tumor formation in heterozygous mTmG 100 days post-EP. Nuclear EGFP+ Rosa26H3f3a-K27M/Pdgfra/Trp53 cells form a large striatal tumor. Inset D-1 shows a lack of significant cortical infiltration. E) A littermate Rosa26H3f3aG34R/Pdgfra/Trp53 exhibits a glial hyperplasia in the striatum and cortex but no tumor is evident. F) K27M tumor at 120 days post-EP is predominantly sub-cortical. G) Cortically-infiltrating G34R tumor at 120 days post-EP. H-I) Confocal pathology of K27M tumor at low mag (H), and high mag (I). J) Low mag pathology of G34R tumor. K) Comparison of survival across H3.3. groups (wildtype-blue, K27M-green, and G34R-red) all containing Pdgfra D842V and Trp53 R270H. L) Chart of the site of K27M versus G34R tumors. *Because of the later onset of tumor growth in G34R groups and their inconsistent survival times, we were unable to collect 2 of 7 G34R samples before death to definitively ascertain initial tumor site. M-N) Experimental schematic for co-EP of K27M and G34R plasmids O-P) G34R and K27M immunostaining of co-EPed tumors in sequential sections. (smFP-myc shown in insets). Q) Quantification of normalized cell counts from tumor

Article Snippet: plasmid: MADR pDonor-smFP-Myc (dark) WPRE , This paper , Addgene Plasmid #130987.

Techniques: Plasmid Preparation, Comparison, Immunostaining

Journal: Cell

Article Title: Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

doi: 10.1016/j.cell.2019.08.013

Figure Lengend Snippet: A) Schematic of cell dissociation and scRNA-seq B) UMAP depicting CCA alignment of 3 MADR mouse K27M scRNA-seq datasets from 3 distinct tumors, colored by cluster based on HVG programs P1-4 from (Filbin et al., 2018) C) Heatmap depicting marker genes emerging from unbiased clustering of mouse K27M cells D) Program and expression featureplots from CCA of mouse K27M tumors. E) UMAP depicting CCA alignment of 6 human K27M datasets from 6 distinct tumors (Filbin et al., 2018), colored by cluster F) Heatmap depicting markers genes emerging from unbiased clustering of human K27M cells G) Program and expression featureplots from CCA of human K27M tumors. H) UMAP depicting CCA alignment of 3 MADR mouse K27M datasets and 6 human K27M datasets (Filbin et al., 2018), colored by cluster I) Program and expression featureplots from CCA of combined mouse and human K27M tumors. J) UMAP depicting CCA alignment of 9 K27M datasets from the mouse and human brain colored by sample K) Heatmap using gene list from (Filbin et al., 2018) demonstrates a high concordance of gene expression between murine and human K27M glioma cells. L) scRNA-seq derived proliferation metrics are comparable across mouse and human sample

Article Snippet: plasmid: MADR pDonor-smFP-Myc (dark) WPRE , This paper , Addgene Plasmid #130987.

Techniques: Marker, Expressing, Gene Expression, Derivative Assay

Journal: Cell

Article Title: Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

doi: 10.1016/j.cell.2019.08.013

Figure Lengend Snippet: A) Flp-Cre vector catalyzes either Cre-mediated excision or dRMCE on Rosa26mTmG allele in the presence of a MADR donor vector, resulting in two distinct recombinant products. B) Nucleofection of heterozygous Rosa26WT/mTmG mNSCs result in three possible lineages: tdTomato+, EGFP+, and TagBFP2+. C) Live imaging of representative cells with non-overlapping fluorescent colors. Scale bars, 100μm D) Schematic of cell preparation for single-cell western blot. E) Frequency of fluorescence intensities comparing MADR and PiggyBac transgenic cells. F) Representative examples of single-cell western blots for PiggyBac and MADR groups. (Note that this is not a pure population and so some cells express the Histone H3 loading control protein but no TagBFP2. Also, many lanes are empty as is typical for this assay). G) MADR-compatible TRE-smFP plasmids for MADR MAX. H) Dox induces efficient smFP expression allowing for orthogonal imaging of 4 independent reporters in vitro. Scale bar, 100μm I) High magnification confocal z-section demonstrates that each cell expresses a single smFP reporter. Scale bar, 10μm J) Schematic of AAVS1 locus targeting for HUMAN MADR by TALEN or CRISPR/Cas9 K) HEK293T cells containing AAVS1-targeted MADR recipient site expressing tdTomato and TagBFP2-V5-nls Scale bar, 100μm L) MADR-HEK293T cells transfected with MADR pDonor smFP-myc (Bright) or TagBFP-3XFlag showing GFP or BFP autofluorescence among non-inserted tdTomato+ cells. Scale bar, 100μm M) High mag image of cells from L exhibiting tdTomato and smFP-myc in a mutually exclusive manner. Scale bar, 10μm

Article Snippet: plasmid: MADR pDonor-smFP-Myc (dark) WPRE , This paper , Addgene Plasmid #130987.

Techniques: Plasmid Preparation, Recombinant, Imaging, Single Cell Western, Fluorescence, Transgenic Assay, Control, Expressing, In Vitro, CRISPR, Transfection

Journal: Cell

Article Title: Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

doi: 10.1016/j.cell.2019.08.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: plasmid: MADR pDonor-smFP-Myc (dark) WPRE , This paper , Addgene Plasmid #130987.

Techniques: Virus, Cloning, Recombinant, Lysis, Library Quantification, Sequencing, Plasmid Preparation, Mutagenesis, Software

Journal: Cell

Article Title: Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

doi: 10.1016/j.cell.2019.08.013

Figure Lengend Snippet: A) Standard postnatal EP protocol targeting the ventricular zone in P2 heterozygous Rosa26WT/mTmG pups with DNA mixture of a Flp-Cre vector and a donor plasmid B) Postnatal EP recapitulates in vitro nucleofection experiment and yields TagBFP2+ MADR along with EGFP+ and tdTomato+ lineages at 2 weeks post-EP. Scale bar, 100μm C) Different concentrations of recombinase and donor plasmids result in various efficiencies of both MADR and Cre-excision recombination reactions in vivo. All mixtures contained a nuclear TagBfp2 reporter plasmid. (See Supp. Fig. 2D for representative images from this quantitation). Error bars indicate standard error of the mean (SEM). D) Schematic of plasmid delivery for combinatorial MADR MAX “brainbow” like multiplex labeling E) Low mag image of olfactory bulb displaying multiplex smFP-based MADR MAX EPed cells and immunostaining for the smFP-linked epitope tags. Scale bar, 100μm F) High mag image of cells from E exhibiting expression of a single smFP epitope tag per neuron. Scale bar, 10μm G) Schematic of expansion microscopy and brightfield image example H) MADR pDonor smFP-myc sh.Nf1 miR-E plasmid for simultaneous knockdown of Nf1 and smFP-myc labeling of transgenic cells I) Image of EPed striatum showing two populations of reporter labeled cells—EGFP and smFP-myc (i.e. Nf1 knockdown cells). J) Pre-expansion smFP-myc cell body K) Post-expansion of cell in J L) Post-expansion EGFP astrocyte displaying “super-resolution” detail. M) Schematic of MADR pDonor-TagBFP2-P2A-VCre and FlEx VCre reporter plasmids for mosaic analysis with tertiary recombinase N) EPed striatum with FlpO-2A-Cre, MADR pDonor-TagBFP2, HypBase and FlEx VCre reporter. Scale bar, 50μm O) Striatum of littermate of mouse shown in N with FlpO-2A-Cre, MADR pDonor-TagBFP2–2A-VCre, HypBase and FlEx VCre reporter exhibiting VCre-dependent FlEx reporter (smFP-myc). Scale bar, 50μm

Article Snippet: plasmid: MADR pDonor-smFP-HA (dark) WPRE , This paper , Addgene Plasmid #131007.

Techniques: Plasmid Preparation, In Vitro, In Vivo, Quantitation Assay, Multiplex Assay, Labeling, Immunostaining, Expressing, Microscopy, Knockdown, Transgenic Assay

Journal: Cell

Article Title: Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

doi: 10.1016/j.cell.2019.08.013

Figure Lengend Snippet: A) Donor construct for miR-E shRNAs against Nf1, Pten, and Trp53 tied to TagBFP2 reporter B) Validation of knockdown efficacy of multi-miR-E function by qPCR. C) 6-month-old mouse sagittal section showing a hyperplasia of TagBFP2+ cells but no tumor. Scale bar, 1mm D) Plasmid for MADR of a TagBFP2-V5 reporter protein and SpCas9 E) Sequencing of TdTomato-/EGFP- glioma cells exhibit InDels in Nf1 and Trp53. F) MADR insertion of TagBFP2-V5 reporter and Cas9 with co-EPed PCR-derived sgRNAs yields high grade glioma observable through labeling of 3 genetic reporter-defined populations in a coronal section of both hemispheres. Scale bar, 1000μm G) Plasmid for MADR of an smFP-myc reporter protein and FNLS Cas9n base editor. H) sgRNA-targeting sites (green letters) induce C->T base conversion (red lowercase ‘c’ are targeted) to produce premature stop codons in Nf1, Trp53, and Pten. I) MADR insertion of myc reporter and FNLS Cas9n with co-EPed PCR-derived sgRNAs yields observable expansion of OPC progenitors at two months post-EP through labeling of three genetic reporter-defined populations in a coronal section. Scale bar, 1000μm J) High magnification tdTomato (1), EGFP (2), and Myc tag (3) image showing myc+ populations. Scale bar, 100μm

Article Snippet: plasmid: MADR pDonor-smFP-HA (dark) WPRE , This paper , Addgene Plasmid #131007.

Techniques: Construct, Biomarker Discovery, Knockdown, Plasmid Preparation, Sequencing, Derivative Assay, Labeling

Journal: Cell

Article Title: Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

doi: 10.1016/j.cell.2019.08.013

Figure Lengend Snippet: A-B) Schematic for in utero EP of MADR into E14.5 RCE +/− dams C) In utero EP in RCE mice with HrasG12V oncogene produces mosaic patches of TagBFP+ astrocytes Rosa26HrasG12 but not evidence of invasive glioma D) Schematic of possible outcomes after MADR in homozygous mt/mg recipient mice E) P2 EP of homozygous mt/mg mice with TagBFP2-HrasG12V oncogene F) Postnatal EP in homozygous Rosa26mTmG P2 pups with HrasG12V oncogene produces two different tumor types (Blue-only Rosa26HrasG12V×2 and blue-and-green Rosa26HrasG12V×1) Scale bars: 2mm G) Representative tumor formation in homozygous mTmG 3 months post-EP. Blue-only Rosa26HrasG12V×2 cells occupy a larger section of the tumor than blue-and-green Rosa26HrasG12V×1, correlating with phosphor-Rb1 protein expression. Scale bars: 1mm H) Zoom-in images of regions 1 and 2 from G show phosphorylated-Rb1 expression correlates largely with blue-only cells. Scale bars: 50μm I) Plasmid schematics for expression of ependymoma-associated fusion proteins J) Stitch of YAP1-MAML1D; p16/p19 Cas9 targeting induced ependymoma-like tumor. K) Survival analysis of Ependymoma MADR model mice L) Ependymoma-like tumor in a 3-month-old C11orf95-RELA; p16/p19 Cas9-targeted mouse

Article Snippet: plasmid: MADR pDonor-smFP-HA (dark) WPRE , This paper , Addgene Plasmid #131007.

Techniques: In Utero, Expressing, Plasmid Preparation

Journal: Cell

Article Title: Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

doi: 10.1016/j.cell.2019.08.013

Figure Lengend Snippet: A) Schematic of donor plasmid for MADR with multiple recurrent pediatric glioma driver mutations B) Schematic of the plasmid delivery and electrode sweep employed to target striatal and cortical germinal niches simultaneously C) Zoomed view from B showing the respective cortical (magenta) and striatal (orange) germinal niches that are targeted D) Representative tumor formation in heterozygous mTmG 100 days post-EP. Nuclear EGFP+ Rosa26H3f3a-K27M/Pdgfra/Trp53 cells form a large striatal tumor. Inset D-1 shows a lack of significant cortical infiltration. E) A littermate Rosa26H3f3aG34R/Pdgfra/Trp53 exhibits a glial hyperplasia in the striatum and cortex but no tumor is evident. F) K27M tumor at 120 days post-EP is predominantly sub-cortical. G) Cortically-infiltrating G34R tumor at 120 days post-EP. H-I) Confocal pathology of K27M tumor at low mag (H), and high mag (I). J) Low mag pathology of G34R tumor. K) Comparison of survival across H3.3. groups (wildtype-blue, K27M-green, and G34R-red) all containing Pdgfra D842V and Trp53 R270H. L) Chart of the site of K27M versus G34R tumors. *Because of the later onset of tumor growth in G34R groups and their inconsistent survival times, we were unable to collect 2 of 7 G34R samples before death to definitively ascertain initial tumor site. M-N) Experimental schematic for co-EP of K27M and G34R plasmids O-P) G34R and K27M immunostaining of co-EPed tumors in sequential sections. (smFP-myc shown in insets). Q) Quantification of normalized cell counts from tumor

Article Snippet: plasmid: MADR pDonor-smFP-HA (dark) WPRE , This paper , Addgene Plasmid #131007.

Techniques: Plasmid Preparation, Comparison, Immunostaining

Journal: Cell

Article Title: Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

doi: 10.1016/j.cell.2019.08.013

Figure Lengend Snippet: A) Schematic of cell dissociation and scRNA-seq B) UMAP depicting CCA alignment of 3 MADR mouse K27M scRNA-seq datasets from 3 distinct tumors, colored by cluster based on HVG programs P1-4 from (Filbin et al., 2018) C) Heatmap depicting marker genes emerging from unbiased clustering of mouse K27M cells D) Program and expression featureplots from CCA of mouse K27M tumors. E) UMAP depicting CCA alignment of 6 human K27M datasets from 6 distinct tumors (Filbin et al., 2018), colored by cluster F) Heatmap depicting markers genes emerging from unbiased clustering of human K27M cells G) Program and expression featureplots from CCA of human K27M tumors. H) UMAP depicting CCA alignment of 3 MADR mouse K27M datasets and 6 human K27M datasets (Filbin et al., 2018), colored by cluster I) Program and expression featureplots from CCA of combined mouse and human K27M tumors. J) UMAP depicting CCA alignment of 9 K27M datasets from the mouse and human brain colored by sample K) Heatmap using gene list from (Filbin et al., 2018) demonstrates a high concordance of gene expression between murine and human K27M glioma cells. L) scRNA-seq derived proliferation metrics are comparable across mouse and human sample

Article Snippet: plasmid: MADR pDonor-smFP-HA (dark) WPRE , This paper , Addgene Plasmid #131007.

Techniques: Marker, Expressing, Gene Expression, Derivative Assay

Journal: Cell

Article Title: Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

doi: 10.1016/j.cell.2019.08.013

Figure Lengend Snippet: A) Flp-Cre vector catalyzes either Cre-mediated excision or dRMCE on Rosa26mTmG allele in the presence of a MADR donor vector, resulting in two distinct recombinant products. B) Nucleofection of heterozygous Rosa26WT/mTmG mNSCs result in three possible lineages: tdTomato+, EGFP+, and TagBFP2+. C) Live imaging of representative cells with non-overlapping fluorescent colors. Scale bars, 100μm D) Schematic of cell preparation for single-cell western blot. E) Frequency of fluorescence intensities comparing MADR and PiggyBac transgenic cells. F) Representative examples of single-cell western blots for PiggyBac and MADR groups. (Note that this is not a pure population and so some cells express the Histone H3 loading control protein but no TagBFP2. Also, many lanes are empty as is typical for this assay). G) MADR-compatible TRE-smFP plasmids for MADR MAX. H) Dox induces efficient smFP expression allowing for orthogonal imaging of 4 independent reporters in vitro. Scale bar, 100μm I) High magnification confocal z-section demonstrates that each cell expresses a single smFP reporter. Scale bar, 10μm J) Schematic of AAVS1 locus targeting for HUMAN MADR by TALEN or CRISPR/Cas9 K) HEK293T cells containing AAVS1-targeted MADR recipient site expressing tdTomato and TagBFP2-V5-nls Scale bar, 100μm L) MADR-HEK293T cells transfected with MADR pDonor smFP-myc (Bright) or TagBFP-3XFlag showing GFP or BFP autofluorescence among non-inserted tdTomato+ cells. Scale bar, 100μm M) High mag image of cells from L exhibiting tdTomato and smFP-myc in a mutually exclusive manner. Scale bar, 10μm

Article Snippet: plasmid: MADR pDonor-smFP-HA (dark) WPRE , This paper , Addgene Plasmid #131007.

Techniques: Plasmid Preparation, Recombinant, Imaging, Single Cell Western, Fluorescence, Transgenic Assay, Control, Expressing, In Vitro, CRISPR, Transfection

Journal: Cell

Article Title: Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

doi: 10.1016/j.cell.2019.08.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: plasmid: MADR pDonor-smFP-HA (dark) WPRE , This paper , Addgene Plasmid #131007.

Techniques: Virus, Cloning, Recombinant, Lysis, Library Quantification, Sequencing, Plasmid Preparation, Mutagenesis, Software

Journal: Cell

Article Title: Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

doi: 10.1016/j.cell.2019.08.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: plasmid: MADR pDonor-smFP-V5 (dark) WPRE , This paper , Addgene Plasmid #131006.

Techniques: Virus, Cloning, Recombinant, Lysis, Library Quantification, Sequencing, Plasmid Preparation, Mutagenesis, Software

Journal: Cell

Article Title: Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

doi: 10.1016/j.cell.2019.08.013

Figure Lengend Snippet: A) Standard postnatal EP protocol targeting the ventricular zone in P2 heterozygous Rosa26WT/mTmG pups with DNA mixture of a Flp-Cre vector and a donor plasmid B) Postnatal EP recapitulates in vitro nucleofection experiment and yields TagBFP2+ MADR along with EGFP+ and tdTomato+ lineages at 2 weeks post-EP. Scale bar, 100μm C) Different concentrations of recombinase and donor plasmids result in various efficiencies of both MADR and Cre-excision recombination reactions in vivo. All mixtures contained a nuclear TagBfp2 reporter plasmid. (See Supp. Fig. 2D for representative images from this quantitation). Error bars indicate standard error of the mean (SEM). D) Schematic of plasmid delivery for combinatorial MADR MAX “brainbow” like multiplex labeling E) Low mag image of olfactory bulb displaying multiplex smFP-based MADR MAX EPed cells and immunostaining for the smFP-linked epitope tags. Scale bar, 100μm F) High mag image of cells from E exhibiting expression of a single smFP epitope tag per neuron. Scale bar, 10μm G) Schematic of expansion microscopy and brightfield image example H) MADR pDonor smFP-myc sh.Nf1 miR-E plasmid for simultaneous knockdown of Nf1 and smFP-myc labeling of transgenic cells I) Image of EPed striatum showing two populations of reporter labeled cells—EGFP and smFP-myc (i.e. Nf1 knockdown cells). J) Pre-expansion smFP-myc cell body K) Post-expansion of cell in J L) Post-expansion EGFP astrocyte displaying “super-resolution” detail. M) Schematic of MADR pDonor-TagBFP2-P2A-VCre and FlEx VCre reporter plasmids for mosaic analysis with tertiary recombinase N) EPed striatum with FlpO-2A-Cre, MADR pDonor-TagBFP2, HypBase and FlEx VCre reporter. Scale bar, 50μm O) Striatum of littermate of mouse shown in N with FlpO-2A-Cre, MADR pDonor-TagBFP2–2A-VCre, HypBase and FlEx VCre reporter exhibiting VCre-dependent FlEx reporter (smFP-myc). Scale bar, 50μm

Article Snippet: plasmid: MADR pDonor-smFP-Flag (dark) WPRE , This paper , Addgene Plasmid #131005.

Techniques: Plasmid Preparation, In Vitro, In Vivo, Quantitation Assay, Multiplex Assay, Labeling, Immunostaining, Expressing, Microscopy, Knockdown, Transgenic Assay

Journal: Cell

Article Title: Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

doi: 10.1016/j.cell.2019.08.013

Figure Lengend Snippet: A) Donor construct for miR-E shRNAs against Nf1, Pten, and Trp53 tied to TagBFP2 reporter B) Validation of knockdown efficacy of multi-miR-E function by qPCR. C) 6-month-old mouse sagittal section showing a hyperplasia of TagBFP2+ cells but no tumor. Scale bar, 1mm D) Plasmid for MADR of a TagBFP2-V5 reporter protein and SpCas9 E) Sequencing of TdTomato-/EGFP- glioma cells exhibit InDels in Nf1 and Trp53. F) MADR insertion of TagBFP2-V5 reporter and Cas9 with co-EPed PCR-derived sgRNAs yields high grade glioma observable through labeling of 3 genetic reporter-defined populations in a coronal section of both hemispheres. Scale bar, 1000μm G) Plasmid for MADR of an smFP-myc reporter protein and FNLS Cas9n base editor. H) sgRNA-targeting sites (green letters) induce C->T base conversion (red lowercase ‘c’ are targeted) to produce premature stop codons in Nf1, Trp53, and Pten. I) MADR insertion of myc reporter and FNLS Cas9n with co-EPed PCR-derived sgRNAs yields observable expansion of OPC progenitors at two months post-EP through labeling of three genetic reporter-defined populations in a coronal section. Scale bar, 1000μm J) High magnification tdTomato (1), EGFP (2), and Myc tag (3) image showing myc+ populations. Scale bar, 100μm

Article Snippet: plasmid: MADR pDonor-smFP-Flag (dark) WPRE , This paper , Addgene Plasmid #131005.

Techniques: Construct, Biomarker Discovery, Knockdown, Plasmid Preparation, Sequencing, Derivative Assay, Labeling

Journal: Cell

Article Title: Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

doi: 10.1016/j.cell.2019.08.013

Figure Lengend Snippet: A-B) Schematic for in utero EP of MADR into E14.5 RCE +/− dams C) In utero EP in RCE mice with HrasG12V oncogene produces mosaic patches of TagBFP+ astrocytes Rosa26HrasG12 but not evidence of invasive glioma D) Schematic of possible outcomes after MADR in homozygous mt/mg recipient mice E) P2 EP of homozygous mt/mg mice with TagBFP2-HrasG12V oncogene F) Postnatal EP in homozygous Rosa26mTmG P2 pups with HrasG12V oncogene produces two different tumor types (Blue-only Rosa26HrasG12V×2 and blue-and-green Rosa26HrasG12V×1) Scale bars: 2mm G) Representative tumor formation in homozygous mTmG 3 months post-EP. Blue-only Rosa26HrasG12V×2 cells occupy a larger section of the tumor than blue-and-green Rosa26HrasG12V×1, correlating with phosphor-Rb1 protein expression. Scale bars: 1mm H) Zoom-in images of regions 1 and 2 from G show phosphorylated-Rb1 expression correlates largely with blue-only cells. Scale bars: 50μm I) Plasmid schematics for expression of ependymoma-associated fusion proteins J) Stitch of YAP1-MAML1D; p16/p19 Cas9 targeting induced ependymoma-like tumor. K) Survival analysis of Ependymoma MADR model mice L) Ependymoma-like tumor in a 3-month-old C11orf95-RELA; p16/p19 Cas9-targeted mouse

Article Snippet: plasmid: MADR pDonor-smFP-Flag (dark) WPRE , This paper , Addgene Plasmid #131005.

Techniques: In Utero, Expressing, Plasmid Preparation

Journal: Cell

Article Title: Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

doi: 10.1016/j.cell.2019.08.013

Figure Lengend Snippet: A) Schematic of donor plasmid for MADR with multiple recurrent pediatric glioma driver mutations B) Schematic of the plasmid delivery and electrode sweep employed to target striatal and cortical germinal niches simultaneously C) Zoomed view from B showing the respective cortical (magenta) and striatal (orange) germinal niches that are targeted D) Representative tumor formation in heterozygous mTmG 100 days post-EP. Nuclear EGFP+ Rosa26H3f3a-K27M/Pdgfra/Trp53 cells form a large striatal tumor. Inset D-1 shows a lack of significant cortical infiltration. E) A littermate Rosa26H3f3aG34R/Pdgfra/Trp53 exhibits a glial hyperplasia in the striatum and cortex but no tumor is evident. F) K27M tumor at 120 days post-EP is predominantly sub-cortical. G) Cortically-infiltrating G34R tumor at 120 days post-EP. H-I) Confocal pathology of K27M tumor at low mag (H), and high mag (I). J) Low mag pathology of G34R tumor. K) Comparison of survival across H3.3. groups (wildtype-blue, K27M-green, and G34R-red) all containing Pdgfra D842V and Trp53 R270H. L) Chart of the site of K27M versus G34R tumors. *Because of the later onset of tumor growth in G34R groups and their inconsistent survival times, we were unable to collect 2 of 7 G34R samples before death to definitively ascertain initial tumor site. M-N) Experimental schematic for co-EP of K27M and G34R plasmids O-P) G34R and K27M immunostaining of co-EPed tumors in sequential sections. (smFP-myc shown in insets). Q) Quantification of normalized cell counts from tumor

Article Snippet: plasmid: MADR pDonor-smFP-Flag (dark) WPRE , This paper , Addgene Plasmid #131005.

Techniques: Plasmid Preparation, Comparison, Immunostaining

Journal: Cell

Article Title: Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

doi: 10.1016/j.cell.2019.08.013

Figure Lengend Snippet: A) Schematic of cell dissociation and scRNA-seq B) UMAP depicting CCA alignment of 3 MADR mouse K27M scRNA-seq datasets from 3 distinct tumors, colored by cluster based on HVG programs P1-4 from (Filbin et al., 2018) C) Heatmap depicting marker genes emerging from unbiased clustering of mouse K27M cells D) Program and expression featureplots from CCA of mouse K27M tumors. E) UMAP depicting CCA alignment of 6 human K27M datasets from 6 distinct tumors (Filbin et al., 2018), colored by cluster F) Heatmap depicting markers genes emerging from unbiased clustering of human K27M cells G) Program and expression featureplots from CCA of human K27M tumors. H) UMAP depicting CCA alignment of 3 MADR mouse K27M datasets and 6 human K27M datasets (Filbin et al., 2018), colored by cluster I) Program and expression featureplots from CCA of combined mouse and human K27M tumors. J) UMAP depicting CCA alignment of 9 K27M datasets from the mouse and human brain colored by sample K) Heatmap using gene list from (Filbin et al., 2018) demonstrates a high concordance of gene expression between murine and human K27M glioma cells. L) scRNA-seq derived proliferation metrics are comparable across mouse and human sample

Article Snippet: plasmid: MADR pDonor-smFP-Flag (dark) WPRE , This paper , Addgene Plasmid #131005.

Techniques: Marker, Expressing, Gene Expression, Derivative Assay

Journal: Cell

Article Title: Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

doi: 10.1016/j.cell.2019.08.013

Figure Lengend Snippet: A) Flp-Cre vector catalyzes either Cre-mediated excision or dRMCE on Rosa26mTmG allele in the presence of a MADR donor vector, resulting in two distinct recombinant products. B) Nucleofection of heterozygous Rosa26WT/mTmG mNSCs result in three possible lineages: tdTomato+, EGFP+, and TagBFP2+. C) Live imaging of representative cells with non-overlapping fluorescent colors. Scale bars, 100μm D) Schematic of cell preparation for single-cell western blot. E) Frequency of fluorescence intensities comparing MADR and PiggyBac transgenic cells. F) Representative examples of single-cell western blots for PiggyBac and MADR groups. (Note that this is not a pure population and so some cells express the Histone H3 loading control protein but no TagBFP2. Also, many lanes are empty as is typical for this assay). G) MADR-compatible TRE-smFP plasmids for MADR MAX. H) Dox induces efficient smFP expression allowing for orthogonal imaging of 4 independent reporters in vitro. Scale bar, 100μm I) High magnification confocal z-section demonstrates that each cell expresses a single smFP reporter. Scale bar, 10μm J) Schematic of AAVS1 locus targeting for HUMAN MADR by TALEN or CRISPR/Cas9 K) HEK293T cells containing AAVS1-targeted MADR recipient site expressing tdTomato and TagBFP2-V5-nls Scale bar, 100μm L) MADR-HEK293T cells transfected with MADR pDonor smFP-myc (Bright) or TagBFP-3XFlag showing GFP or BFP autofluorescence among non-inserted tdTomato+ cells. Scale bar, 100μm M) High mag image of cells from L exhibiting tdTomato and smFP-myc in a mutually exclusive manner. Scale bar, 10μm

Article Snippet: plasmid: MADR pDonor-smFP-Flag (dark) WPRE , This paper , Addgene Plasmid #131005.

Techniques: Plasmid Preparation, Recombinant, Imaging, Single Cell Western, Fluorescence, Transgenic Assay, Control, Expressing, In Vitro, CRISPR, Transfection

Journal: Cell

Article Title: Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

doi: 10.1016/j.cell.2019.08.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: plasmid: MADR pDonor-smFP-Flag (dark) WPRE , This paper , Addgene Plasmid #131005.

Techniques: Virus, Cloning, Recombinant, Lysis, Library Quantification, Sequencing, Plasmid Preparation, Mutagenesis, Software