title: Quality improvement of transgenic cloned bovine embryos using an aggregation method: Effects on cell number, cell ratio, embryo perimeter, mitochondrial distribution, and gene expression profile.
abstract: The production of cloned embryos using conventional methods has extremely low success rates owing to low embryo quality. To improve the quality of cloned bovine embryos expressing enhanced green fluorescent protein (EGFP), we applied an aggregation culture method. The EGFP gene was transfected into bovine fetal fibroblasts using a retroviral vector system. Somatic cell nuclear transfer was performed using these cells, and the resulting embryos were cultured in aggregates or individually. Gene expression was analyzed by a microarray, and differentially expressed genes were validated by quantitative real-time polymerase chain reaction. The total number of cells per blastocyst and the ratio of inner cell mass cells to trophectoderm cells were higher in aggregated transgenic cloned blastocysts (agBL; 368.7 ± 109.6 and 1:4.8, respectively) than in in vitro-fertilized blastocysts (ivfBL; 189.8 ± 65.8 and 1:2.6, respectively) and nonaggregated transgenic cloned blastocysts (sBL; 113.1 ± 36.3 and 1:1.5, respectively; P < 0.05 and P < 0.01, respectively). Moreover, the blastocyst perimeter was larger in the agBL group than in the ivfBL and sBL groups (1168.8 ± 200.23 vs. 887.33 ± 187.62 and 678 ± 226.1 μm; P < 0.05). In addition, mitochondrial fluorescence intensity was higher in the agBL group than in the ivfBL and sBL groups (P < 0.05). The number of apoptotic cells per blastocyst was lower in the ivfBL and agBL groups than in the sBL group (3.7 ± 2.2 and 3.4 ± 2.1 vs. 6.7 ± 6.8; P < 0.05). The genes identified in the microarray belonged to 18 categories. Expression of the Krüppel-like factor 4 gene, which is associated with cell proliferation, development, and transcription, was 7.2-fold higher in the agBL group than in the ivfBL group (P < 0.05) but did not differ between the sBL and ivfBL groups (P > 0.05). Expression of the heat shock 70-kDa protein 1A gene, which is associated with apoptosis, was 12-fold higher in the sBL group than in the ivfBL and agBL groups (P < 0.05). Expression of a stemness-related gene (octamer-binding transcription factor 4) and trophectoderm-specific genes (homeobox protein CDX2 and keratin 18) was higher in the agBL group than in the sBL group (P < 0.05). However, expression of the stemness gene homeobox protein NANOG did not differ among the groups (P > 0.05). Taken together, these data suggest that the aggregation method improves the quality of cloned embryos expressing EGFP and might be helpful in animal cloning.
authors.affiliation: Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, Republic of Korea; Department of Animal Science, Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju, Republic of Korea.
authors.forename: J I
authors.initials: JI
authors.lastname: Bang
authors.affiliation: Department of Animal Science, Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju, Republic of Korea.
authors.forename: J I
authors.initials: JI
authors.lastname: Jin
authors.affiliation: Animal Production Department, Faculty of Agriculture, Cairo University, Giza, Egypt.
authors.forename: N
authors.initials: N
authors.lastname: Ghanem
authors.affiliation: Department of Animal Science, Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju, Republic of Korea.
authors.forename: B H
authors.initials: BH
authors.lastname: Choi
authors.affiliation: Department of Animal Science, Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju, Republic of Korea.
authors.forename: M
authors.initials: M
authors.lastname: Fakruzzaman
authors.affiliation: Department of Animal Science, Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju, Republic of Korea.
authors.forename: A N
authors.initials: AN
authors.lastname: Ha
authors.affiliation: Department of Animal Science, Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju, Republic of Korea.
authors.forename: K L
authors.initials: KL
authors.lastname: Lee
authors.affiliation: Department of Animal Science and Biotechnology, Industrial Education Field, Sangji Youngseo College, Wonju, South Korea.
authors.forename: S J
authors.initials: SJ
authors.lastname: Uhm
authors.affiliation: Department of Animal Science and Biotechnology, Industrial Education Field, Sangji Youngseo College, Wonju, South Korea.
authors.forename: D H
authors.initials: DH
authors.lastname: Ko
authors.affiliation: Department of Physiology, Catholic University of Daegu School of Medicine, Daegu, Republic of Korea.
authors.forename: B C
authors.initials: BC
authors.lastname: Koo
authors.affiliation: Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, Republic of Korea; Department of Animal Science, Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju, Republic of Korea.
authors.forename: J G
authors.initials: JG
authors.lastname: Lee
authors.affiliation: Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, Republic of Korea; Department of Animal Science, Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju, Republic of Korea. Electronic address: ikong7900@gmail.com.
authors.forename: I K
authors.initials: IK
authors.lastname: Kong
mesh_terms: D000818:Animals; D015415:Biomarkers; D001755:Blastocyst / Q000378:metabolism*; D002417:Cattle / Q000196:embryology*; D002449:Cell Aggregation; D019976:Cloning, Organism; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000502:physiology*; D030781:Organisms, Genetically Modified; D059467:Transcriptome / Q000502:physiology*
chemical_list: D015415:Biomarkers; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Aggregation method; Bovine; IVF; Microarray; Quantitative real-time PCR; SCNT