Fig 1: Establishment of GHR-modified single-cell colonies. a Schematic diagram of DNA deletion mediated by the dual-sgRNAs/Cas9 system. The brown line indicates the GHR gene structure from the Ensmbl database. “<” indicates the direction of transcription. The red and green arrows indicate the targeting sites of the designed sgRNAs. The blue arrows indicate cleavage sites mediated by the sgRNAs/Cas9 system. The orange segment and line indicate the deleted 47-bp DNA fragment mediated by the dual-sgRNAs/Cas9 system. WT: wild-type allele. C3: single-cell colony #3. b PCR products harboring the targeted GHR region amplified from pig single-cell colonies (M, DNA maker DL2000; C, colony). c Detection of sgRNAs/Cas9-mediated on-target GHR cleavage by the T7ENI cleavage assay. The band size of wild-type PCR product is 777 bp, and the sizes of T7ENI cut bands are about 555 and 175 bp. The mismatched about 47 bp band was not detected. d Sequence of modified GHR in donor cells for SCNT. The red font indicates the sgRNA1 targeting site, and the green font indicates the sgRNA2 targeting site. C6: single-cell colony #6. “–”: deletion; N/N: positive sequencing out of total sequencing
Fig 2: Germline transmission of GHR-modified alleles. a Picture of partial GHRKO F1 pigs; the leftmost pig is wild-type. b Picture of GHRKO F2 pigs. c PCR products harboring the targeted GHR region amplified from GHRKO F1 pigs. d Detection of the GHR genotype by the T7ENI cleavage assay in GHRKO F1 pigs (M, DNA maker DL2000; WT, PCR product amplified from wild-type pig digested by T7ENI). e Sequences of modified GHR detected in GHRKO F1 and F2 pigs
Fig 3: Highly efficient DNA deletion mediated by dual-sgRNAs/Cas9. a Detected modified GHR sequences of 40 alleles in 20 single-cell colonies. The five types of mutations detected include - 47, - 46, - 49, - 18 and - 1 bp. WT: wild-type sequence. “–”: deletion. N/40: Modified GHR alleles detected out of 40 total alleles. b Percentage of GHR mutation types detected in 40 alleles. c Percentage of DNA fragment deletion. d Detection of sgRNA1 and sgRNA2 programmed Cas9 targeting effect by T7ENI cleavage assay. e The mutant genotype of GHR mediated by sgRNA1/Cas9 or sgRNA2/Cas9 system. The red font indicates the sgRNA1 targeting site, and the green font indicates the sgRNA2 targeting site
Fig 4: Detection of dual-sgRNAs/Cas9-mediated GHR modification in piglets. a, b Picture of GHRKO pigs showing their small stature and normal fertility. c Picture of the live GHRKO fetus that was used to establish cell lines for recloning. d PCR products harboring the targeted region of GHR amplified from selected piglets. e Detection of dual-RNAs/Cas9-mediated on-target GHR cleavage by the T7ENI cleavage assay in piglets. P1–P3 piglets were cloned from the C3 single-cell colony; P4–P7 piglets were cloned from the C6 single-cell colony. (M, DNA maker DL2000; C, colony; WT, wild-type PCR product digested by T7ENI). f Modified GHR sequences detected in piglets and fetuses. F1 and F2 indicate the two fetuses cloned from the C6 cell colony. P4–P15 were cloned piglets from C6 cell colony
Fig 5: Evaluation of GHR expression in GHRKO pigs. a Immunofluorescence analysis of GHR in fibroblasts isolated from GHRKO piglets. Fibroblasts were stained with anti-GHR secondary antibodies (red). DAPI (blue) staining indicates the nucleus. The data are representative of at least three independent experiments. b Relative expression of GHR mRNA in tissues. Relative GHR expression was detected in heart, liver, kidney and muscle tissues. GAPDH served as the internal control. The data were derived from four GHRKO pigs and three wildtype pigs; the bars represent the mean ± SEM; *P < 0.05 and **P < 0.01. c Immunoblotting analysis of porcine GHR in heart and kidney tissues. Protein extracted from wild-type pig was used as the control. ß-Actin was used as the internal control. d Immunochemical analysis of various tissues from GHRKO pigs. Heart, liver, kidney, spleen, testis and muscle tissues from GHRKO pigs were GHR-deficient. Wild-type pigs were used as the control. e Compared to those in control pigs, serum IGF-I and blood glucose levels were significantly decreased in GHRKO pigs, which was consistent with that observed in Laron patients; **P < 0.01
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