Journal: PLoS ONE
Article Title: Human PPP1R26P1 Functions as cis-Repressive Element in Mouse Rb1
Figure Lengend Snippet: Targeting human PPP1R26P1 into mouse Rb1 intron 2. A) Top: structure of the human PPP1R26 gene on chromosome 9, consisting of four exons; non-coding sequences are indicated by lower box height. Four small CpG islands are represented by black lines below exon 4. Middle: mRNA structure of PPP1R26 . Below: scheme of exons 1 to 3 (black vertical boxes) of the human RB1 locus on chromosome 13. Integration of PPP1R26P1 in intron 2 of RB1 occurred in inverted orientation and skipping part of exon 1 of the PPP1R26 mRNA. The pseudogene has two larger CpG islands, CpG42 and CpG85, their methylation status is indicated by circles: filled – methylated, open – not methylated. Expression of RB1 is skewed in favor of the maternal allele indicated by a thicker arrow. The new exon 2B is shown as white box in PPP1R26P1 , it splices onto exon 3 of RB1 . The light gray boxes in intron 2 indicate ECRs A and B. B) Generation of modified ES cells. The order of targeting experiments is shown, indicating the number and designations of clones analyzed. Above the arrows the type of experiment is given: SNV or PPP1R26P1 construct: introduction of targeting vectors; PFGE: analysis of phasing of the two targeting events; Cre: removal of selection cassette by Cre expression. C) The targeting strategy in murine ES cells. Top: wild type mouse Rb1 locus on chromosome 14 is shown from exon 1 to 5 (black boxes), also showing the positions of ECRs A and B (grey boxes). Below: constructs used to introduce the SNV (indicated by P for the newly generated PstI restriction site) into exon 3 of Rb1 (SNV targeting vector) and to introduce human PPP1R26P1 (gray dotted box, positions of CpG42 and CpG85 shown as black lines) into intron 2 of Rb1 ( PPP1R26P1 targeting vector). The homology region of the latter was amplified by PCR, primers are indicated as black arrows. Both contracts contained a neomycin selection cassette (striped box) flanked by either loxP (white triangles) or loxP511 (black triangles) sites, respectively. Restriction enzymes: B: BamHI, E: EcoRI, H: HindIII, Hc: HincII, P: PstI, Sw: SwaI, X: XcmI. Southern blot probes are indicated in the top scheme as black lines and labeled. The two possible genomic combinations of targeted alleles are depicted in the lower panel: genotype SNV_ PPP1R26P1 /wt, having both targeting events and the same allele or genotype SNV / PPP1R26P1 with the two targeting events on different alleles. D) Southern blot analysis of targeted clones carrying the SNV in exon 3 of Rb1 . Probe SNP 5’ ext hybridized to HincII-digested genomic DNA shows an 8 kb fragment for the targeted allele in Rb1 _SNVneo and a 12 kb band for the targeted allele after selection cassette removal in Rb1 _SNV clones. E) Southern blot analysis for targeting of PPP1R26P1 into intron 2 of Rb1 . Probe KIAA 5’ ext hybridized to HincII-digested genomic DNA identifies a 4 kb fragment for the targeted allele in the SNV_ PPP1R26P1 neo clone and a 10kb fragment in SNV_ PPP1R26P1 clones after removal of the selection cassette. F) Southern blot analysis of PFGE to determine phasing of the two targeting events. Probe KIAA 5’ ext was hybridised to EcoRV-digested DNA to identify either a 23 kb and a 27 kb band in clones having genotype SNV_ PPP1R26P1 neo/wt (both events on one allele) or a 20kb and a 30kb fragment in clones with genotype SNV / PPP1R26P1 neo with the two events occurred on different alleles.
Article Snippet: PPP1R26P1 was generated by long-range PCR using the Phusion Flash high-fidelity PCR master mix (Thermo Scientific) using BAC RPCIB753O20259Q (obtained from imaGenes) as template and introducing SwaI restriction sites for cloning into pACYC_D4_ECR and the introduction of the neomycin selection cassette.
Techniques: Methylation, Expressing, Modification, Clone Assay, Construct, Selection, Introduce, Generated, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Southern Blot, Labeling