[보고서]한국 오계의 생산성 향상을 위한 유용 유전자 발굴 및 활용기술 개발

박태섭

보고서 정보

주관연구기관

서울대학교
Seoul National University

연구책임자

박태섭

참여연구자

김시원 , 이정효 , 허유경 , 김은지

보고서유형

최종보고서

발행국가

대한민국

언어

한국어

발행년월

2018-02

과제시작연도

2017

주관부처

농촌진흥청
Rural Development Administration(RDA)

연구관리전문기관

농촌진흥청
Rural Development Administration

등록번호

TRKO201800043331

과제고유번호

1395049224

사업명

차세대바이오그린21

DB 구축일자

2019-05-04

키워드

오계.생식세포.산육성.유전자교정.유전체선발.Oge.Germ cell.Productivity.Genome editing.Genetic selection.

DOI

https://doi.org/10.23000/TRKO201800043331

표 Chicken myostatin (MSTN) gene structure and targeted site design for nickase-mediated mutant induction. (A) Sequences of chicken MSTN exon1 and intron. The bold ATG indicates the start codon and the blue italic sequences present the forward and reverse primer for PCR amplification. The red sequences are the targeted sites of the Cas9-D10A nickase. (B) The left and right target sequences (highlighted boxes) for the nickase. The target site has +7bp offset and consequently, the targeted sites show 5’ overhanging 43bp after the nickase-mediated DNA breaks. The red sequences are the targeted sites and the blue sequences are the complimentary sequences. The bold sequences indicate the protospacer adjacent motif (PAM). The arrow heads are the cleavage positions by the Cas-D10A nickase
표 Knockout of chicken myostatin (MSTN) gene and identification of mutant genotype in the targeted sites. (A) Fluorescence-activated cell sorting (FACS) of GFP-positive cells after co-transfection of the Cas9-D10A nickase expression vector with green fluorescent gene (GFP) gene and targeted multiplex guide RNAs (gRNAs). (B) Phenotypic comparison between regular and MSTN knockout DF1 cells. (C) Mutant genotypes of the targeted MSTN gene induced by the nickase. (D) Western blotting of chicken MSTN in regular and MSTN knockout DF1 cells
표 List of primer sequences for chicken myostatin (MSTN) gene and the predicted off-target sites for left and right guide RNA (gRNA) sequences
표 Analysis of the predicted off-target sites. (A) Sequence analysis of the predicted off-target for right guide RNA (gRNA) sequences. (B) Sequence analysis of the predicted off-target for left gRNA sequences. Parentheses indicate single nucleotide polymorphism (SNP) in the targeted sites
표 MyoD gene knockout in QM7 cells. (A) Genomic sequence and structure of the quail MyoD gene. (B) Expression vector of Cas9-GFP and MyoD guide RNA (gRNA). The U6 promoter controls gRNA transcription followed by a termination signal. (C) Experimental overview and fluorescence-activated cell sorting (FACS) for the development of single cell-derived MyoD knockout QM7 sublines. (D) Genotypes of MyoD-knockout mixed QM7 cells. (E) Genotypes of single cell-derived MyoD-knockout QM7 sublines (QM7 MyoD KO#4)
표 Genotyping analysis of MyoD gene knockout QM7 cells. (A) Genotypes of MyoD-knockout mixed QM7 cells. (B) Genotypes of single cell-derived MyoD-knockout QM7 sublines (QM7 MyoD KO #4)
표 Characterization of MyoD-knockout QM7 cells. (A) Pax7 expression in regular QM7 (rQM7) and MyoD-knockout QM7 (MyoD KO#4) cells. There were no significant differences between rQM7 and MyoD KO#4 cells. (B) Morphology of undifferentiated rQM7 and MyoD KO#4 cells. (C) Morphological changes 3 or 6 days after differentiation. rQM7 cells transformed into myotubes during differentiation. No morphological changes were evident in MyoD KO#4 cells after 6 days of differentiation (scale bars=200um)
표 (A) Nuclear fusion in regular QM7 (rQM7) and MyoD-knockout QM7 (MyoD KO#4) cells during myotube differentiation. No fusion was evident in MyoD KO#4 cells even after 6 days of differentiation. (B) Western blotting detected MyoD protein in rQM7 and MyoD KO#4 cells after 3 days of differentiation. (C) RT-PCR analysis of myogenic differentiation-related genes and quantitative RT-PCR of the MyoD gene in rQM7 and MyoD KO#4 cells during differentiation. (D) Detection of myogenic differentiation-related genes in rQM7 and MyoD KO#4 cells during differentiation (scale bars=200um)
표 String analysis of differentially expressed genes (DEGs) processed by RNA-sequencing data. (A) String analysis of total DEGs of rQM7 and MyoD KO#4 cells during differentiation. (B) String analysis of DEGs significantly decreased in MyoD KO#4 cells during differentiation. The red box indicates the MyoD transcript
표 Sequencing analysis of genomic PCR and RT-PCR for quail SH3RF2 gene structure
표 The gRNA-Cas9 expression vector. Expression vector and Cas9-green fluorescent protein (GFP) and qSH3RF2 gRNA sequences. The U6 promoter controls gRNA transcription followed by a termination signal
표 Genomic mutation analysis in single cell-derived knockout quail myoblast (QM7) cells. (A) T7E1 assay for the targeted sites in quail SH3RF2 in knockout (KO) QM7 cells. (B) Mutated target DNA sequences of SH3RF2 KO QM7#1 and #4 cells. The dashed and red sequences denote the deleted nucleotides and targeted sequences, respectively. SH3RF2 KO QM7#4 cells exhibited the mutant genotype (61 and 155 nucleotide deletions), whereas SH3RF2 KO QM7#1 cells exhibited a mixed genotype (15, 33, and 70 nucleotide deletions)
표 Differentiation and characterization of SH3RF2 KO QM7#4 cells. (A) Morphology of undifferentiated rQM7 and SH3RF2 KO QM7#4 cells (scale bar = 200 μm). (B) Quantitative reverse transcription-polymerase chain reaction of undifferentiated rQM7 and SH3RF2 KO QM7#4 cells (left panel) and Western blot analysis of MyoD expression in rQM7 and SH3RF2 KO QM7#4 cells 6 days after differentiation (right panel). (C) Morphological changes 6 days after differentiation. Similar to rQM7 cells, SH3RF2 KO QM7#4 cells transformed into myotubes during differentiation. Arrows indicate nuclear fusion and multiple nuclei in the differentiated myotubes (scale bar = 200 μm). (D) Comparison of the differentiated areas between rQM7 and SH3RF2 KO QM7#4 cells 3 days after the induction of differentiation. KO, knockout; QM7, quail myoblast; rQM7, regular QM7
표 String analysis of differentially expressed genes (DEGs) processed by RNA-Seq data. (A) String interaction analysis of the SH3RF2 gene in undifferentiated QM7 cells. (B) String analysis of DEGs in SH3RF2 KO QM7#4 cells during differentiation. The red box indicates the SH3RF2 transcript. No statistically significant interaction or regulatory pathway during myotube differentiation was identified. SH3RF2, SH3 domain containing ring finger 2; KO, knockout; QM7, quail myoblast
표 Morphology of growing chicken embryonic myoblast cells, pCM cells
표 Proliferation assay of reg. pCM by WST-1 reagent
표 (A) Differentiated pCM myoblast into myotubes in the differentiation media for 4 days. (B) Detection of nucleus fusion in the differentiated myotubes of pCM cells with DAPI staining
표 miRNA expression vector for chicken Foxo3 gene Knockdown and targeted miRNA sequences. miRNA expression vector was constructed into piggyBac transposon elements and basic miRNA scaffold sequences were originated from chicken endogeneous miRNA126
표 Establishment of Foxo3 Knockdown (KD) pCM subline after transfecton of miRNA expression vector and puromycin treatment (10ug/ml). Foxo3 KD pCM cells are stably expressing the eGFP and it could be expected to co-express the miRNAs for Foxo3 transcript
표 Comparison of gene expression profiles (Foxo3, Pax7, SRF, and CEBPB) between reg. pCM and Foxo3 KD pCM by Quantitative RT-PCR (qRT-PCR) analysis
표 Comparison of Foxo3 expression between reg. pCM and Foxo3 KD pCM by Western blotting
표 Comparison of proliferation capacity between reg. pCM and Foxo3 KD pCM by WST-1 reagent
표 (A) Differentiated Foxo3 KD pCM cells into myotubes in the differentiation media for 4 days. (B) Detection of nucleus fusion in the differentiated myotubes of Foxo3 KD pCM cells with DAPI staining
표 Characterization and comparisons of myotube differentiation between Reg. pCM and Foxo3 KD pCM cells. (A) Percentage comparison of differentiated area after 4 day differentiation. (B) Width/length ratios of differentiated area after 4 day differentiation. (C) Comparison of nuclei numbers per differentiated area after 4 day differentiation
표 RT-PCR analysis of CXCR4 transcript in different chicken cell types (chicken PGCs, DT40, and DF1). PGCs and DT40 cells strongly expressed CXCR4, but DF1 cells did not
표 (A) Schematic clustered regularly interspaced short palindromic repeats (CRISPR) designs for the chicken CXCR4 gene on chromosome 7 and the genomic mutation analysis. The sequences of the guiding RNA target site for CRISPR CXCR4 were located in the second exon of the chicken CXCR4 gene. The TGG in red is a protospacer adjacent motif. The lowercase and capital letters are the sequences of an intron and exon, respectively. (B) The mutated target DNA sequences of the chicken PGC sublines (CXCR4 KO PGC #2 and #3) induced by CRISPR CXCR4. Dashes in the DNA sequences denote the deleted nucleotides; the inserted nucleotide in CXCR4 KO PGC# 2 is indicated by a lowercase letter in italics. (C) The mutant genotypes show the frameshift in the translated proteins that induce amino acid changes and a stop codon (asterisk)
표 (A) Transgene expression vectors for enhanced green fluorescent protein (eGFP), Discosoma red fluorescent protein (DsRed), or the chicken CXCR4 gene controlled by a cytomegalovirus (CMV) promoter. The constructs were inserted between the 5’-terminal repeat (5’-TR) and the 3’-TR piggyBac transposon elements. The neomycin-resistant (NeoR) gene was used as a selection marker. (B) RT-PCR analysis after transfection and selection of piggyBac CMV-chicken CXCR4 overexpression. (C) Fluorescent images of eGFP or DsRed-expressing normal PGCs and eGFP-expressing CXCR4 KO PGCs. All PGC sublines strongly expressed the fluorescent reporter transgene (magnification, 100×). (D) eGFP-expressing DF1 and DT40 cells after transfection and selection of the piggyBac CMV-eGFP expression vector (magnification, 100×)
표 In vivo migration assay of normal PGCs and CXCR4 KO PGCs after transplantation into recipient embryos. (A) Dissected embryonic gonads on Day 6 of incubation after transferring a combination of normal and CXCR4 KO PGCs. A combination of normal GFP and DsRed PGCs was used as a control, and the migration capacity of the eGFP-expressing CXCR4 KO PGCs was compared after co-injection of normal DsRed-expressing PGCs. An equal number of cells (1,000:1,000) was mixed and injected into the blood vessel of recipient embryos (magnification, 100×). (B) A comparison of the total number of migrated PGCs in the recipient embryonic gonads on Day 6. Each dot indicates the sum of the migrated PGC number in the right and left gonads of each individual recipient embryo. (C) A comparison of the number of migrated PGCs in the right and left gonads of the recipient embryos on Day 6. Each dot indicates the number of the individual recipient embryonic right or left gonad
표 In vivo migration assay of normal PGCs and CXCR4 KO PGCs after transplantation into recipient embryos. (A) Dissected embryonic gonads on Day 10 of incubation after transfer of the normal and CXCR4 KO PGC combination. A combination of the normal GFP and DsRed PGCs was used as a control, and the migration capacity of eGFP-expressing CXCR4 KO PGCs was compared after co-injection of normal DsRed-expressing PGCs. An equal number of cells (1,000:1,000) was mixed and injected into the blood vessel of recipient embryos (magnification of the second panel, 50×; all other panels, 100×). (B) Dissected embryonic gonads on Day 6 of incubation after transferring a combination of CXCR4 KO PGCs and normal PGCs (10,000:1,000) (magnification of the second panel, 50×; all other panels, 100×)
표 In vivo migration assay of GFP-expressing DT40 and CXCR4 OE DF1 cells. GFP-expressing DT40 or DF1 cells carrying the CMV-controlled chicken CXCR4-overexpressing transgene were transplanted into the blood vessel of recipient embryos. The embryonic gonads were dissected on Days 6 (A) and 10 (B). GFP-expressing cells were detected using a fluorescent microscope
표 Genomic structure of chicken SH3RF2 gene and sequence alignment of White Leghorn (WL) SH3RF2 gene to examine the exon4 genome sequences
표 Sequence informatin of targeted gRNA primers and in chicken SH3RF2 gene (A) and T7E1 assay of SH3RF2 KO DF1 cells to detected the induced mutation. The arrows indicate the digested fragments from the heteroduplex PCR product
표 Detection of the induced mutants of SH3RF2 in chicken primordial germ cells through T7E1 assay and genotyping. The efficiency of mutation was 33.3% and the genotypes were 4nt, 5nt, 6nt deletion types. The red letters and lines are the targeted sequences and the deleted nucleotides, respectively. The bold letters of PAM sequences are CGG
표 Controlled inducible transgene expression in chicken DF1 and quail QM7 cells. (A) Leaky expression of the GFP transgene is regulated by the cumate promoter. In DF1 cells, there is no GFP expression in the absence of cumate (upper panel), but slightly leaky GFP expression was apparent in QM7 cells. (B) Flow cytometric analysis of GFP-expressing cells among cumate-GFP DF1 and QM7 cells in the off condition (in the absence of cumate). (C) The cumate-GFP cells were cultured in the presence of cumate for 3 or 6 days. All figures show phase contrast (left) and fluorescent (right) images (Scale bar=100 um)
표 Flow cytometry following induction of the GFP transgene by different concentrations of cumate. Flow cytometry determination of the percentages of GFP-positive cells after cumate induction in cumate-GFP DF1 (A) and cumate-GFP QM7 (B) cells. The different superscripts next to the percentages of GFP-positive cells indicate statistically significant differences between treatments (P < 0.05)
표 Quantitative RT-PCR analysis in cumate-MyoD DF1 cells following cumate induction. The expression of MyoD (A), myogenin (B), and desmin (C) are expressed relative to ß-actin expression. The different superscripts of the relative expression for each transcript indicate statistically significant differences between treatments (P < 0.05)
표 Schematic diagram of expression vector for transgene excision with Cre recombinase. loxP71 sequences were used for Cre recombinase recognition and CMV promoter was designed for transgene expression. mCherry could not be expressed without transgene recombination because of stop codon of eGFP and ployA signal. 3’-TR and 5’-TR were piggyBac transposon elements and the arrows indicate the primer positions for PCR detection
표 Transgene excision analysis in loxP-eGFP-mCherry expression vector-transfected DF1 cells after transfection of Cre recombinase expression vector. (A) eGFP-expressing DF1 cells after transfection of loxP-eGFP-mCherry expression vector and G418-selection (upper panel). 7 days after re-transfection of Cre recombinase expression vector into the loxP-eGFP-mCherry-transfected cells, GFP-negative cells (P5 cell population) were sorted out by FACS (lower panel). (B) PCR analysis of the loxP-eGFP-mCherry-transfected cells and eGFP-negative cell population derived by Cre recombination. (C) Sequence analysis after excision of eGFP transgene with Cre recombinase. eGFP transgene located between loxP71 elements was exactly excised by Cre recombinase but mCherry transgene could not be expressed due to the frameshift of the open reading frame induced by ATG sequences (dotted box) prior to mCherry transgene start codon
표 Schematic diagram of expression vector for transgene translocation with Cre recombinase. loxP66 and loxP71 sequences were used for Cre recombinase recognition and CMV promoter was designed for transgene expression. eGFP transgene was replaced with DsRed transgene after Cre recombination
표 Transgene translocation analysis in loxP66-eGFP-loxP71 expression vector-transfected DF1 cells after co-transfection with Cre recombinase expression vector and loxP66-DsRed-loxP71 vector. Translocation analysis of transgene expression in loxP66-eGFP-loxP71 expression vector-transfected cells after 14 days of co-transfection with Cre recombinase expression vector and loxP66-DsRed-loxP71 vector. After 14 days of co-transfection, any cell did not overlap the expression of both eGFP and DsRed indicating that eGFP transgene was excised and DsRed transgene was replaced by Cre recombination
표 기초 오계 계군 체중 분포 (270일령 기준) 및 선발 조건
표 Primarily cultured Korean Oge primordial germ cells (PGCs) derived from embryonic gonads at 6days (left panel) and the established Korean Oge PGC line (right panel). (mgnification; 100x)
표 Genomic DNAs from whole blood cells of Korean Oge chickens (high weight males and low weight males) and Cornish chickens
표 Validation of the constructed library with chicken genomic DNA and estimation of library sizes by electropherogram
표 Summary of NGS data from the constructed library
표 Genomic variation analysis between three chicken lines (White Leghorn, Cornish and Korean Oge)
표 (A) GFP expression in quail blood cells isolated from blood vessel of 50-hr-old embryos after lipofection with the different concentrations of piggyBacCMV-GFP and transposase DNAs (magnification; 10×). (B) Comparison of lipofection efficiencies in quail blood cells with the different amounts of plasmid DNAs by flow analysis. The ratio of piggyBacCMV-GFP and transposase plasmid was 1:1 (w:w) in all treatments. Three separate experiments were replicated and statistically analysed (p<0.005) (NS; no significant difference)
표 GFP detection in quail tissues after direct in ovo lipofection (DiL). Mixture of piggyBacCMV-GFP and transposase plasmid with liposome was transferred into blood vessel at 50-hr-old quail embryos. After injection of DNA-liposome complex, the GFP expre- ssion was detected in heart, kidney, liver and stomach at 12 days of incubation under a confocal laser scanning microscope
표 GFP detection in quail tissues after direct in ovo lipofection (DiL). Mixture of piggyBacCMV-GFP and transposase plasmid with liposome was transferred into blood vessel at 50-hr-old quail embryos. After injection of DNA-liposome complex, the GFP expression was detected in recipient embryonic gonads at 12 days of incubation under a confocal laser scanning microscope

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[국가R&D연구보고서] 한국 오계의 생산성 향상을 위한 유용 유전자 발굴 및 활용기술 개발
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[국가R&D연구보고서] 한국 오계의 생산성 향상을 위한 유용 유전자 발굴 및 활용기술 개발 Development of systemic breeding module to improve the economic productivity of Korean Oge 원문보기

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[국가R&D연구보고서] 한국 오계의 생산성 향상을 위한 유용 유전자 발굴 및 활용기술 개발
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