Journal: Genome Research

Article Title: Cre-dependent Cas9-expressing pigs enable efficient in vivo genome editing

doi: 10.1101/gr.222521.117

Figure Lengend Snippet: Generation and characterization of Cre-dependent Cas9-expressing pigs. ( A ) A diagram for TALEN-mediated knock-in of Cre-dependent Cas9-expressing cassette into the p Rosa26 locus. Gray triangles, wild-type lox P site; white triangles, mutant lox P2272 site; SA, splice acceptor; TALEN target site and PCR primers ( F1 , R1 , F2 , R2, F, and R ) are indicated. ( B,C ) Schematic of two alternative patterns of Cre-mediated activation of SpCas9 and tdTomato: ( B , left ) Cre recombinase induces inversion of both Neo and iCas9 expression cassettes flanked by two lox P sites, followed by excision of Neo expression cassette flanked by two lox P2272 sites ( C ); ( B , right ) Cre recombinase-induced inversion of iCas9 expression cassettes by two lox P2272 sites, followed by excision of Neo expression cassette between two lox P sites ( C ). After inversion of iCas9 expression cassette and removal of the Neo expression cassette, SpCas9 and tdTomato expression are controlled by the endogenous porcine Rosa26 promoter ( C ). ( D ) Morphologically normal piglets were born from SCNT with the p Rosa 26-iCas9 PFFs. ( E ) PCR analysis confirmed the correct homologous recombination at the p Rosa26 locus in 3/5 cloned piglets. Three positive piglets were all monoallelic modifications, as detected by PCR (F2 + F + R), consistent with those of cells chosen as nuclear donors. Primer pairs are shown in A and in Supplemental Table 3 . ( F ) SpCas9 and tdTomato activations using Cre recombinase in fibroblasts isolated from the ear tissues of cloned piglets shown in D . Cells were infected with Cre-EGFP lentivirus, and the expression of tdTomato and EGFP were observed after 48 h by using a fluorescence microscope. Scale bars, 50 µm. ( G ) FACS analysis of Cre recombinase-induced tdTomato activation in p Rosa26 -iCas9 fibroblasts. ( H ) Western blot analysis was used to directly verify SpCas9 expression in p Rosa26 -iCas9 fibroblasts infected with lentivirus containing Cre. Cells not infected with Cre lentiviruses and WT cells were used as negative control. ( I ) H&E staining of the lung, liver, kidney, heart, and spleen of sacrificed wild-type and Cre-dependent Cas9-expressing piglets.

Article Snippet: A pFlexibleDT-p Rosa26 -iCas9 targeting vector was constructed on the basis of the pFlexibleDT-p Rosa26 -iEGFP targeting vector (Addgene; #60952) that we reported previously ( ).

Techniques: Expressing, Knock-In, Mutagenesis, Activation Assay, Homologous Recombination, Clone Assay, Isolation, Infection, Fluorescence, Microscopy, Western Blot, Negative Control, Staining

Journal: Genome Research

Article Title: Cre-dependent Cas9-expressing pigs enable efficient in vivo genome editing

doi: 10.1101/gr.222521.117

Figure Lengend Snippet: Ex vivo single- and multigene knockout in p Rosa26 -iCas9 fibroblasts. ( A ) Schematic diagram of ex vivo genome editing experimental workflow. First, p Rosa26 -iCas9 fibroblasts were isolated from the ear tissues of Cre-dependent Cas9-expressing pigs; second, the isolated p Rosa26 -iCas9 fibroblasts were infected with lentivirus containing Cre, EGFP, and specific sgRNAs; finally, the genome modifications in infected cells were analyzed at 1 wk posttransduction. ( B ) Design of sgRNA targeting porcine GGTA1 locus and three representative Sanger sequencing reads of subclones into T-vector from p Rosa26 -iCas9 fibroblasts. ( C ) A diagram of lentiviral vectors for Cre recombinase, EGFP, and GGTA1 -sgRNA expression. ( D ) Sanger sequencing of PCR products containing GGTA1- sgRNA targeting site. Upper : p Rosa26 -iCas9 fibroblasts uninfected with lentivirus; bottom : p Rosa26 -iCas9 fibroblasts infected with lentivirus containing Cre recombinase, EGFP, and GGTA1 -sgRNA. ( E ) GGTA1- sgRNA-mediated cleavage in wild-type and p Rosa26 -iCas9 fibroblasts infected or uninfected with lentivirus was analyzed by using a T7EN1 cleavage assay. ( F ) Western blot analysis for verifying α-Gal epitope and SpCas9 expression in wild-type and p Rosa26 -iCas9 fibroblasts infected or uninfected with lentivirus. Beta actin was used as a control. ( G ) Design of sgRNAs targeting early exons of porcine APC , BRCA 1, or BRCA2 , and three representative Sanger sequencing reads of subclones into T-vector from p Rosa26 -iCas9 fibroblasts infected with lentivirus AB12. ( H ) A diagram of lentiviral vector AB12 containing Cre recombinase, EGFP , APC -sgRNA, BRCA1 -sgRNA, and BRCA2 -sgRNA. ( I ) Sanger sequencing results of PCR products containing APC- sgRNA, BRCA1- sgRNA, and BRCA2- sgRNA targeting sites.

Article Snippet: A pFlexibleDT-p Rosa26 -iCas9 targeting vector was constructed on the basis of the pFlexibleDT-p Rosa26 -iEGFP targeting vector (Addgene; #60952) that we reported previously ( ).

Techniques: Ex Vivo, Knock-Out, Isolation, Expressing, Infection, Sequencing, Plasmid Preparation, Cleavage Assay, Western Blot

Journal: Genome Research

Article Title: Cre-dependent Cas9-expressing pigs enable efficient in vivo genome editing

doi: 10.1101/gr.222521.117

Figure Lengend Snippet: Induction of EML4–ALK rearrangements in p Rosa26 -iCas9 fibroblasts. ( A ) Schematic representation of porcine EML4–ALK rearrangements induced by CRISPR-Cas9. EML4 -sgRNA and ALK -sgRNA (red) were designed to target the mutation sites of the porcine EML4 gene intron 14 and porcine ALK gene intron 13. PCR primers are indicated (primers A, B, C, and D). ( B ) PCRs were performed to analyze ALK–EML4 (primers A and D were used) and EML4–ALK rearrangements (primers B and C were used) and large fragment deletion (primers B and D were used). The fragment amplified by primers A and B was used as positive control ( bottom panel). ( C ) The ALK–EML4 and EML4–ALK PCR products were subcloned into T-vector, and the Sanger sequencing results of five independent clones and a representative chromatogram are shown in the left and right panels, respectively. ( D ) Diagram of EML4–ALK mRNA fusion transcripts ( upper panel). Agarose gel electrophoresis analysis suggested that the RT-PCR products of EML4–ALK mRNA fusion transcripts only exist in p Rosa26 -iCas9 fibroblasts infected with both EML4 -sgRNA and ALK -sgRNA; GAPDH was used as positive control ( bottom panel). ( E ) The Sanger sequencing results of RT-PCR products showing that the sequences of EML4–ALK mRNA fusion transcripts are identical with predicted sequences ( bottom panel).

Article Snippet: A pFlexibleDT-p Rosa26 -iCas9 targeting vector was constructed on the basis of the pFlexibleDT-p Rosa26 -iEGFP targeting vector (Addgene; #60952) that we reported previously ( ).

Techniques: CRISPR, Mutagenesis, Amplification, Positive Control, Plasmid Preparation, Sequencing, Clone Assay, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Infection

Journal: Genome Research

Article Title: Cre-dependent Cas9-expressing pigs enable efficient in vivo genome editing

doi: 10.1101/gr.222521.117

Figure Lengend Snippet: Establishment and characterization of 4-OHT-inducible system in p Rosa26 -iCas9 fibroblasts. ( A ) Schematic of 4-OHT-induced SpCas9 and tdTomato expression in p Rosa26- iCas9 fibroblasts infected with lentivirus containing CreERT2. ( B ) Percentage of EGFP- and tdTomato-positive cells under different concentrations of 4-OHT (0–10 µM). ( C ) Western blot analysis for verifying SpCas9 expression with different concentrations of 4-OHT inductions. ( D ) T7EN1 assays showing indel formation at the GGTA1 locus in p Rosa26- iCas9 infected with lentivirus containing CreERT2, EGFP, and GGTA1 -sgRNA and simultaneously supplied with 4-OHT, while not in uninfected or untreated fibroblasts. ( E ) Sanger sequencing analysis of the GGTA1-sgRNA targeting site. Top: p Rosa26- iCas9 fibroblasts; middle , p Rosa26- iCas9 fibroblasts infected with lentivirus containing CreERT2 and GGTA1 -sgRNA, but not supplied with 4-OHT; bottom , p Rosa26- iCas9 fibroblasts infected with lentivirus containing CreERT2 and GGTA1 -sgRNA, simultaneously supplied with 4-OHT.

Article Snippet: A pFlexibleDT-p Rosa26 -iCas9 targeting vector was constructed on the basis of the pFlexibleDT-p Rosa26 -iEGFP targeting vector (Addgene; #60952) that we reported previously ( ).

Techniques: Expressing, Infection, Western Blot, Sequencing

Journal: Nature Communications

Article Title: Editing DNA methylation in vivo

doi: 10.1038/s41467-025-67222-5

Figure Lengend Snippet: A Schematic representation of the Lox-Stop-Lox-dCas9-DNMT3A-P2A-GFP (LSL-dC9-D) transgene cassette inserted at the Rosa26 locus. pCAG cytomegalovirus enhancer fused with chicken beta-actin promoter and rabbit beta-globin splice acceptor, LSL Lox-stop-lox cassette, NLS nuclear localization sequence, P2A porcine teschivoris-1 2A self-cleaving sequence, eGFP enhanced green fluorescent protein, WPRE woodchuck hepatitis virus posttranscriptional regulatory element, bGHpA bovine growth hormone polyadenylation signal. B Western blot of DNMT3A, dCas9, and Tubulin expressions in brain tissue isolated from LSL-dCas9-DNMT3A-GFP mice and LSL-dCas9-DNMT3A-GFP; EIIa-Cre mice. C Immunofluorescent staining of GFP in the hippocampus of LSL-dCas9-DNMT3A-GFP and LSL-dCas9-DNMT3A-GFP; EIIa-Cre mice. Scale bar: 100 μm. D Immunofluorescent staining of DAPI, mCherry, GFP, dCas9 colocalization in mice injected contralaterally with either AAV9-mCherry or AAV9-mCherry-Cre. Scale bar: 100 μm. E Quantification of dCas9-DNMT3A induction efficiency in mCherry and mCherry-Cre labeled cells. ( n = 3 mice per group, two-sided t test, P = 0.000010).

Article Snippet: The targeted KV-1 mESC clones were generated by electroporation of the modified Rosa26 targeting vector construct Ai9 (Addgene 22799) containing a transgenic LSL-dCas9-DNMT3A or LSL-dCas9-TET1 cassette and identification of positive clones by genotyping PCR.

Techniques: Sequencing, Virus, Western Blot, Isolation, Staining, Injection, Labeling

Journal: Nature Communications

Article Title: Editing DNA methylation in vivo

doi: 10.1038/s41467-025-67222-5

Figure Lengend Snippet: A Schematic representation of the Lox-Stop-Lox-dCas9-TET1-P2A-GFP (LSL-dC-T) transgene cassette inserted at the Rosa26 locus. pCAG cytomegalovirus enhancer fused with chicken beta-actin promoter and rabbit beta-globin splice acceptor, LSL Lox-stop-lox cassette, NLS nuclear localization sequence, P2A porcine teschivoris-1 2A self-cleaving sequence, eGFP enhanced green fluorescent protein, WPRE woodchuck hepatitis virus posttranscriptional regulatory element, bGHpA bovine growth hormone polyadenylation signal. B Western blot of GFP, dCas9, and Tubulin expressions in brain tissue isolated from LSL-dCas9-TET1-GFP mice and LSL-dCas9-TET1-GFP; EIIa-Cre mice. C Immunofluorescent staining of GFP in the hippocampus of LSL-dCas9-TET1-GFP and LSL-dCas9-TET1-GFP; EIIa-Cre mice. Scale bar: 100 μm. D Immunofluorescent staining of DAPI, mCherry, eGFP, dCas9 colocalization in mice injected contralaterally with either mCherry or mCherry-Cre. Scale bar: 100 μm. E Quantification of dCas9-TET1 induction efficiency in mCherry-Cre and mCherry-labeled cells. ( n = 3 mice per group, two-sided t test, P = 1.2 × 10 −7 ). F Quantification of the percentage of NeuN+ cells in mCherry− and mCherry+ populations. ( n = 8 mice per group, two-sided t test, P = 2.81 × 10 −8 ).

Article Snippet: The targeted KV-1 mESC clones were generated by electroporation of the modified Rosa26 targeting vector construct Ai9 (Addgene 22799) containing a transgenic LSL-dCas9-DNMT3A or LSL-dCas9-TET1 cassette and identification of positive clones by genotyping PCR.

Techniques: Sequencing, Virus, Western Blot, Isolation, Staining, Injection, Labeling

Journal: Nucleic Acids Research

Article Title: Efficient mouse transgenesis using Gateway-compatible ROSA26 locus targeting vectors and F1 hybrid ES cells

doi: 10.1093/nar/gkp112

Figure Lengend Snippet: Generation and analysis of Gateway-compatible conditional ROSA26-promoter-based expression alleles. ( A ) LR reactions performed between the pROSA26DV-1 vector and cDNA containing pEntry clones for generation of the ROSA26 targeting vector ( B ). S1 and S2 represent sequencing primers. Black and blue rectangles are the 5′, 3′ external and internal DNA probes. The black double-headed arrows show the expected 5.8-kb (wild-type) and 3.0-kb band sizes (targeted) knock-in ( C ) alleles in Southern blot analysis using the 5′ external probe on BamH1-digested genomic DNA. Blue arrow shows the expected 4.0-kb band size (targeted) in EcoRV digests using internal neo probe and the red lines depict the 37-kb (wild-type) and 8.8-kb band size (targeted) in KpnI digests using the external 3′ probe. ( D ) Cre-mediated deletion of the intervening lox P flanked PGK-neo-3xpA (STOP) cassette results in the ROSA26-locus-based expression of an exon1-cDNA-IRES-eGFP bi-cistronic fusion transcript (SA-splice acceptor). ( E ) Example of Southern blot confirmation of 3 PCR positive ROSA26-Snai2 based ES cell clones used to confirm 5′ integration (left), 3′ integration (middle) and single-copy integration (right).

Article Snippet: An additional benefit of using the Gateway recombination system in tandem with our ROSA26 locus targeting vectors is that there is an ever-growing list of Gateway-compatible pEntry clones ( ) as well as commercially and publicly available attB -containing cDNA vectors that can be converted into pEntry clones by performing BP reactions with donor vectors containing the corresponding att P sites (as an example see the following web resource: http://www.imagenes-bio.de/products/sets_libraries/image ).

Techniques: Expressing, Plasmid Preparation, Clone Assay, Sequencing, Knock-In, Southern Blot

Journal: Nucleic Acids Research

Article Title: Efficient mouse transgenesis using Gateway-compatible ROSA26 locus targeting vectors and F1 hybrid ES cells

doi: 10.1093/nar/gkp112

Figure Lengend Snippet: Generation and analysis of ROSA26-promoter-based mice. ( A) Bright field (left panel) and fluorescence (eGFP) images (right panel) of a control non-Cre-excised ROSA26-Snai2-IRES-eGFP Tg/+ embryo (left) and mutant Sox2-Cre Tg/+ , ROSA26-Snai2-IRES-eGFP Tg/+ embryo (right) showing ubiquitous eGFP expression. ( B ) Merged fluorescence (eGFP) and bright field image of Nestin-Cre Tg/+ , ROSA26 Tg/+ embryo showing eGFP expression in brain and neural tube at E10.5. ( C ) Fluorescence image of E17.5 forelimb of a Col2-Cre Tg/+ , ROSA26- Tg/+ embryos showing eGFP expression in forelimb elements. ( D ) Image of totally agouti, seemingly 100% G4 ES-cell-derived G0 male mice. ( E ) Q-RT-PCR analysis performed on two E11.5 wild-type and 3 Sox2-Cre Tg/+ , ROSA26-Snai2-IRES-eGFP Tg/+ mutant littermates showing 2-4-fold upregulated Snai2 mRNA expression levels. ( F ) Wild-type (wt) E14.5 embryo (left) and two mutant (mut) Sox2-Cre Tg/+ , ROSA26-Snai2-IRES-eGFP Tg/+ littermates (right) ubiquitously expressing Snai2 showing pallor and cephalic haemorrhage (arrow). ( G ) Wild-type (wt) control E10.5 embryo compared with two Sox2-Cre Tg/+ , ROSA26-Snai1-IRES-eGFP Tg/+ mutant (mut) littermates ubiquitously expressing Snai1 showing severe developmental defects (left panel).

Article Snippet: An additional benefit of using the Gateway recombination system in tandem with our ROSA26 locus targeting vectors is that there is an ever-growing list of Gateway-compatible pEntry clones ( ) as well as commercially and publicly available attB -containing cDNA vectors that can be converted into pEntry clones by performing BP reactions with donor vectors containing the corresponding att P sites (as an example see the following web resource: http://www.imagenes-bio.de/products/sets_libraries/image ).

Techniques: Fluorescence, Control, Mutagenesis, Expressing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Efficient mouse transgenesis using Gateway-compatible ROSA26 locus targeting vectors and F1 hybrid ES cells

doi: 10.1093/nar/gkp112

Figure Lengend Snippet: Analysis of parental and Cre-excised ROSA26-pCAGG-Snai1 ES cell clones. ( A ) X-gal staining of ES cell colonies of sense (upper-left panel) and anti-sense targeted pCAAG βgeo expressing cells (upper-right panel) (100×). ( B ) Q-RT-PCR and ( C ) western blot analysis showing higher β-galactosidase mRNA and protein levels in the anti-sense clones compared to sense clones. β-Actin loading controls (lowest panel). ( D ) Altered ES cell colony morphology of pCAGG-Snai1 (Cre+) expressing ES cell clones (arrows, top right panel) compared with normal ES cell (Cre–) colony morphology of parental clones (top left panel) (200×). ( E ) Q-RT-PCR analysis showing elevated levels of Snai1 mRNA transcripts in Cre-excised (Cre+) pCAGG-Snai1 cells compared with ROSA26-promoter-driven Snai1 mRNA levels. ( F ) Western blot analysis showing decreased E-Cadherin protein levels in ROSA26-Snai1 and pCAGG-Snai1 Cre-excised (Cre+) ES cells, respectively compared to parental ROSA-Snai1 non-Cre excised (Cre–) E-Cadherin levels (middle panel). β-Actin loading controls (lowest panel).

Article Snippet: An additional benefit of using the Gateway recombination system in tandem with our ROSA26 locus targeting vectors is that there is an ever-growing list of Gateway-compatible pEntry clones ( ) as well as commercially and publicly available attB -containing cDNA vectors that can be converted into pEntry clones by performing BP reactions with donor vectors containing the corresponding att P sites (as an example see the following web resource: http://www.imagenes-bio.de/products/sets_libraries/image ).

Techniques: Clone Assay, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Nucleic Acids Research

Article Title: Efficient mouse transgenesis using Gateway-compatible ROSA26 locus targeting vectors and F1 hybrid ES cells

doi: 10.1093/nar/gkp112

Figure Lengend Snippet: Analysis of parental and Cre-excised ROSA- and pCAGG-promoter-driven Mdm4 ES cells and pCAGG MDM4 mice. ( A ) Q-RT-PCR analysis of Mdm4 mRNA expression levels in parental non-Cre-excised (Cre–) clones compared to enhanced Mdm4 mRNA levels of Cre-excised (Cre+) ROSA26 and pCAGG-promoter-driven samples. ( B ) Immunohistochemical analysis of Sox2-Cre (Cre+) ROSA26-pCAGG-myc-Mdm4 E14.5 tissues using an anti-myc antibody (1/600 dilution) showing widespread cytoplasmic and nuclear localized myc-tagged Mdm4 protein expression in the central nervous system (CNS) and skeletal muscle (red arrow heads) but more mosaic expression in the lung and liver (blue arrow heads) while Cre negative (Cre–) ROSA26-pCAGG-myc-Mdm4 E14.5 tissues show no expression of the myc-tagged Mdm4 protein (400× magnification). Sections were counter-stained with hematoxylin. ( C ) Schematic of parental (top panel) and Cre-excised (middle panel) ROSA26-pCAGG-Mdm4 alleles. PCR analysis of genomic DNA isolated from control non-Cre Tg (Cre–) pCAGG-Mdm4 Tg/+ tail detecting presence of floxed non-recombined allele (G3-G4 primers) but not the excised allele (G5-G4 primers) compared with samples isolated from Sox2-Cre Tg/+ , pCAGG-Mdm4 Tg/+ double transgenic organs (Cre+) showing absence of the floxed allele and only excised allele ( D ) Q-RT-PCR analysis showing widespread and enhanced expression of Mdm4 (grey), myc-tagged Mdm4 (black) and eGFP (white) mRNA transcripts in Sox2-Cre Tg/+ , pCAGG-Mdm4 Tg/+ double transgenic tissues (Cre+) compared with non-excised pCAGG-AS-Mdm4 Tg/+ controls (Cre–). The relative values are set to zero to accommodate for the lack of eGFP and myc-tagged Mdm4 mRNA expression in the absence of Cre-mediated deletion of the floxed β-geo STOP cassette. ( E ) Western blot of tissue lysates from Sox2-Cre Tg/+ , pCAGG-Mdm4 Tg/+ double transgenic mice (Cre+) showing widespread expression of the myc-tagged Mdm4 protein (upper panel) and absence in pCAGG-Mdm4 Tg/+ controls (Cre–). Using an antibody that recognizes both myc-tagged and endogenous Mdm4 protein, the Cre-excised transgenic samples show upregulated myc-tagged Mdm4 protein (*) in all tissues examined compared to endogenous Mdm4 levels. Vinculin protein loading controls (lowest panel).

Article Snippet: An additional benefit of using the Gateway recombination system in tandem with our ROSA26 locus targeting vectors is that there is an ever-growing list of Gateway-compatible pEntry clones ( ) as well as commercially and publicly available attB -containing cDNA vectors that can be converted into pEntry clones by performing BP reactions with donor vectors containing the corresponding att P sites (as an example see the following web resource: http://www.imagenes-bio.de/products/sets_libraries/image ).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Clone Assay, Immunohistochemical staining, Staining, Isolation, Control, Transgenic Assay, Western Blot

Journal: Nucleic Acids Research

Article Title: Efficient mouse transgenesis using Gateway-compatible ROSA26 locus targeting vectors and F1 hybrid ES cells

doi: 10.1093/nar/gkp112

Figure Lengend Snippet: Generation of MultiSite Gateway-compatible ROSA26-targeted pCAGG-promoter-based expression alleles. ( A ) Generation of MultiSite ROSA26 targeting vectors using the 5′-pCAGG- lox P flanked β-geo-3xpA (STOP) cassette, middle-cDNA and 3′-IRES-eGFP reporter pEntry clones. ( B ) In the conditional knock-in alleles before Cre-mediated excision the pCAGG promoter drives the expression of the β-geo (β-galactosidase-neomycin phosphotransferase fusion gene) cassette (arrows) but not downstream cDNA-IRES-eGFP mRNA expression. Shown are the expected fragment lengths using KpnI/EcoRI digests and the 5′ and 3′ as well as internal eGFP probes used to confirm both sense and anti-sense orientation pCAGG-based single-integration transgenes to the ROSA26 locus. ( C ) Following Cre-mediated excision of the floxed β-geo cassette, the pCAGG promoter drives cDNA-IRES-eGFP transgene expression in either a sense or anti-sense orientation relative to the sense ROSA26 promoter.

Article Snippet: An additional benefit of using the Gateway recombination system in tandem with our ROSA26 locus targeting vectors is that there is an ever-growing list of Gateway-compatible pEntry clones ( ) as well as commercially and publicly available attB -containing cDNA vectors that can be converted into pEntry clones by performing BP reactions with donor vectors containing the corresponding att P sites (as an example see the following web resource: http://www.imagenes-bio.de/products/sets_libraries/image ).

Techniques: Expressing, Clone Assay, Knock-In

Journal: bioRxiv

Article Title: Novel EGFRvIII-CAR transgenic mice for rigorous preclinical studies in syngeneic mice

doi: 10.1101/2021.01.31.429020

Figure Lengend Snippet: (A) Schematic representation of the Rosa26 chromosome 6 locus and the location of the targeted insertion. Restriction sites for EcoRI and BglI enzymes and locations for Probes A and B are indicated. (B) Correct genomic targeting of the transgene at the 3' end was confirmed by Southern blotting of BglI-digest genomic DNA with Probe B. (C) Overview of targeting the conditional allele to the Rosa26 locus of C57BL/6J ES cells and generation of founder mice.

Article Snippet: Binding sites for the restriction enzyme AscI were added to the 5’ and 3’ ends of the construct by PCR and used to insert the mCAR sequence into the Rosa26 -targeting vector CTV [(Addgene #15912), ].

Techniques: Southern Blot