title: Generation of five human lactoferrin transgenic cloned goats using fibroblast cells and their methylation status of putative differential methylation regions of IGF2R and H19 imprinted genes.
abstract: BACKGROUND
  Somatic cell nuclear transfer (SCNT) is a promising technique to produce transgenic cloned mammalian, including transgenic goats which may produce Human Lactoferrin (hLF). However, success percentage of SCNT is low, because of gestational and neonatal failure of transgenic embryos. According to the studies on cattle and mice, DNA methylation of some imprinted genes, which plays a vital role in the reprogramming of embryo in NT maybe an underlying mechanism.
  
  
  RESULTS
  Fibroblast cells were derived from the ear of a two-month-old goat. The vector expressing hLF was constructed and transfected into fibroblasts. G418 selection, EGFP expression, PCR, and cell cycle distribution were applied sequentially to select transgenic cells clones. After NT and embryo transfer, five transgenic cloned goats were obtained from 240 cloned transgenic embryos. These transgenic goats were identified by 8 microsatellites genotyping and southern blot. Of the five transgenic goats, 3 were lived after birth, while 2 were dead during gestation. We compared differential methylation regions (DMR) pattern of two paternally imprinted genes (H19 and IGF2R) of the ear tissues from the lived transgenic goats, dead transgenic goats, and control goats from natural reproduction. Hyper-methylation pattern appeared in cloned aborted goats, while methylation status was relatively normal in cloned lived goats compared with normal goats.
  
  
  CONCLUSIONS
  In this study, we generated five hLF transgenic cloned goats by SCNT. This is the first time the DNA methylation of lived and dead transgenic cloned goats was compared. The results demonstrated that the methylation status of DMRs of H19 and IGF2R were different in lived and dead transgenic goats and therefore this may be potentially used to assess the reprogramming status of transgenic cloned goats. Understanding the pattern of gene imprinting may be useful to improve cloning techniques in future.
authors.affiliation: Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing, China ; Human and Animal Physiology, Wageningen University, Wageningen, The Netherlands.
authors.forename: Li
authors.initials: L
authors.lastname: Meng
authors.forename: Yongjie
authors.initials: Y
authors.lastname: Wan
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Sun
authors.forename: Yanli
authors.initials: Y
authors.lastname: Zhang
authors.forename: Ziyu
authors.initials: Z
authors.lastname: Wang
authors.forename: Yang
authors.initials: Y
authors.lastname: Song
authors.forename: Feng
authors.initials: F
authors.lastname: Wang
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D015139:Blotting, Southern; D002417:Cattle; D002453:Cell Cycle; D002478:Cells, Cultured; D019976:Cloning, Organism; D019175:DNA Methylation; D004624:Embryo Transfer; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D018392:Genomic Imprinting; D006041:Goats; D006801:Humans; D007781:Lactoferrin / Q000235:genetics*; D051379:Mice; D053652:Nuclear Transfer Techniques; D016133:Polymerase Chain Reaction; D062085:RNA, Long Noncoding / Q000235:genetics*; D017527:Receptor, IGF Type 2 / Q000235:genetics*
chemical_list: C094335:H19 long non-coding RNA; D062085:RNA, Long Noncoding; D017527:Receptor, IGF Type 2; D007781:Lactoferrin