Fig 1: In vitro transcription and screening of sgRNA.A. Gel electrophoresis of the PCR product of In Vitro Transcription of sgRNA B. PCR product of 819-bp target fragment amplified from the SAT1 gene containing gRNA sequence. C. The PCR amplified SAT1 fragment, the sgRNA, and recombinant Cas9 enzyme were combined in an in vitro cleavage reaction according to the protocol. The reaction mixture was run on 2% Agarose gel. Lane. 1 shows the untreated target SAT1 fragment, and Lane 2 shows the Cas9 treated, cleaved target fragments.
Fig 2: Construction of insert.A. Schematic of the SAT1 locus, indicating exons and introns, the gRNA site location, the mutation sites, the START and STOP codons, and the transcriptional stop AATAAA sequence. Below are the four PCR ‘parts’ and the location of the loxP sites and the primers used for sequence validation. B. The engineered SAT1 insert in pBSSKII+ plasmid with features indicated. C. Schematic of the SAT1 locus after CRISPR/Cas9 mediated incorporation of the target construct. D. Schematic of the SAT1 locus after Cre recombination. BSD = Blasticidin S deaminase; hPGK = human 3-phosphoglycerate kinase promoter.
Fig 3: Validation of clone DM#2.A. Primer pair R15/R19 and R15/R20 amplify the WT or mutant SAT1, respectively. B. Sequence results of the PCR products confirm the conversion of WT codon (AGA) to mutant codon (GCA) of amino acid 101 of SAT1 protein. C. Primer pairs R15/R18 and R17/R18 amplify the WT and mutant SAT1 at nucleotides corresponding to amino acids 101 and 152 of SAT1 protein. D. Sequence results of the PCR products confirm the conversion of WT to mutant SAT1 gene.
Fig 4: Schematic diagram of the mechanism of the protective effect of spermine against high glucose-induced diabetic podocyte injury. ODC, ornithine decarboxylase; SSAT, spermidine/spermine N1-acetyltransferase; Atg5, autophagy protein 5; LC3, microtube-associated proteins 1A/1B light chain 3.
Fig 5: Validation of clone SM#36.A. DNA gel showing DNA amplification of WT or mutant SAT1 using R15/R18 and R17/R18 primers. B. Sequence results of the PCR products confirm the conversion of WT codon (AAG) to mutant codon (AAA) of amino acid 152 of SAT1 protein after Cre recombination. C. DNA gel showing DNA amplification of WT or mutant SAT1 using R15/R18 and R17/R18 primers after Cre recombination. D. Sequence results of the PCR products confirm the conversion of the WT codon (AGA) to the mutant codon (GCA) for amino acid 101; and the WT codon (AAG) to the mutant codon (AAA) for amino acid 152 of SAT1.
Supplier Page from Abcam for Anti-SAT1 antibody