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Fig. 5 | BMC Genomics

Fig. 5

From: Streamlined computational pipeline for genetic background characterization of genetically engineered mice based on next generation sequencing data

Fig. 5

The congenic footprint influences gene expression of Sall2-KO MEFs. a Cartoon comparing a segment of Chr 14 in Sall2 WT versus Sall2-KO MEFs. Grey color denotes the C57BL/6J recipient strain, and the blue segments represent the ESC introgressed genome with corresponding congenic genes, flanking the Sall2 locus (Neo = neomycin cassette used in the homologous recombination). b Validation of several downregulated (left) and upregulated DEGs (right) between Sall2 WT and KO iMEFs. We isolated, reverse transcribed and analyzed RNA from Sall2 WT and KO iMEFs by quantitative real-time PCR. Shown are the expression levels normalized to RNA pol II (Polr2A gene) for every gene when compared to levels in WT. We expressed the values as fold change from WT (N = 3; data is represented as mean ± s.e.m.), blue # P < 0.05; green # P < 0.01; red # P < 0.001 versus WT; Student’s T-test. c Cartoon comparing Chr 14 between scramble (shCtrl) and Sall2-silenced cells (shSall2). Grey color denotes the C57BL/6J recipient strain, showing this comparison as genetically correct. d Left: Representative Western blot for SALL2 and ACTIN in scramble (shCtrl) and Sall2-silenced (shSall2) in WT iMEFs. Right: Cross-validation of genes in Sall2-silenced cells by qPCR as in (B). We normalized the values against RNA pol II (Polr2A gene) and expressed them as fold change from scramble (shCtrl) (N = 3; data is represented as mean ± s.e.m.), blue # P < 0.05; red # P < 0.001 versus shCtrl; Student’s T-test. e Left: IGV snapshot of the WT and congenic Pnp/Pnp2 gene. The numbers depict exons of both genes. Primers used for detection are indicated in every exon (Exon 5 = Forward, Exon 6 = Reverse). We magnified mismatches from IGV in both primers (in red and green), and we underlined it around the reverse primer in exon 6. Right: Schematics of the Pnp gene and the position of primers for quantitative real-time PCR. Lower: Quantitative real-time PCR of Pnp mRNA in Sall2 WT and null iMEFs. Shown are the Pnp expression levels normalized to Polr2A gene for Pnp when compared to levels in WT (N = 4, *** P < 0.001, versus WT; Student’s T-test). f Left: Venn diagram comparing DEGs from Sall2 WT versus KO MEFs (FDR < 0.01, Fold Change > 0.35) with DEGs from a microarray of Sall2 induction in ESCs (Fold Change > 1.3, GSE72350). The overlap reveals 37 common DEGs. Right: DEG cross-validation by fold change from the overlap of both studies. We considered only DEGs with opposing fold changes in both studies. g qPCR validation of two DEGs (Meox1 and Ms4a6d) from the latter in Sall2 WT versus null iMEFs (N = 3; data is represented as mean ± s.e.m.). blue # P < 0.05; green # P < 0.01 versus WT; Student’s T-test. h Cross-validation of Sall2-responsive DEGs in a CRISPR-Cas9 (KO E1A) model in HEK293. Left: Representative Western blot for SALL2 and ACTIN in HEK293 individual clones transfected with Control CRISPR (without sgRNA) or SALL2 E1A CRISPR with the quantification of SALL2 protein bands normalized with ACTIN in the bar graph (N = 4 for control (WT), N = 4 for CRISPR SALL2E1A (KO E1A), ** P < 0.01 versus WT; Student’s T-test.) Right: Validation of several downregulated DEGs in the KO E1A model by qPCR. We expressed the values as fold change from WT (N = 3; data is represented as means±s.e.m.). *** P < 0.001; * P < 0.05; ns, non-significant versus WT; Student’s T-test

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