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

BackgroundSomatic 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.Methodology/Principal FindingsFibroblast 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/SignificanceIn 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.

Abnormal expression of DNA methyltransferases and genomic imprinting in cloned goat fibroblasts

“…H19 expression was decreased, which is in agreement with findings in cloned piglets (Wei et al, 2010). Published studies have revealed that H19 expression was unchanged (Inoue et al, 2002) and that methylation of the H19 DMR resembles that of normal controls in SCNT cloned mice (Han et al, 2008;Shen et al, 2012;Meng et al, 2013). The H19 DMR regulates expression of both H19 and IGF2 (Le et al, 2013); thus, hypermethylation of the H19 DMR would repress H19 expression.…”

“…The H19 DMR regulates expression of both H19 and IGF2 (Le et al, 2013); thus, hypermethylation of the H19 DMR would repress H19 expression. Published studies have revealed that H19 expression was unchanged (Inoue et al, 2002) and that methylation of the H19 DMR resembles that of normal controls in SCNT cloned mice (Han et al, 2008;Shen et al, 2012;Meng et al, 2013). However, in fetuses of ESC cloned mice, H19 was decreased (Humpherys et al, 2001).…”

“…Because cloning efficiency is too low, it is not realistic to use cloned livestock tissues for research. Abnormalities in global methylation patterns (Ohgane et al, 2001;Kremenskoy et al, 2006) and H19 and IGF2R expressions (Yang et al, 2005;Su et al, 2011;Meng et al, 2013) have been described in several cloned species. Thus, ear-derived fibroblast is an excellent resource to study the faulty epigenetic reprogramming.…”

“…Thus, ear-derived fibroblast is an excellent resource to study the faulty epigenetic reprogramming. Abnormalities in global methylation patterns (Ohgane et al, 2001;Kremenskoy et al, 2006) and H19 and IGF2R expressions (Yang et al, 2005;Su et al, 2011;Meng et al, 2013) have been described in several cloned species. However, in goat an understanding of the consequence of SCNT on imprinted gene methylation and on global methylation is lacking.…”

Disease reduction in aquaculture with genetic and genomic technology: current and future approaches

“…Lactoferrin is an 80-kDa protein from the transferrin family that is responsible for binding, transport and metabolism of iron ions (Iyer & Lonnerdal 1993). Lactoferrin has been transferred and expressed in several animal species (Chen et al 2008;Meng et al 2013;Cooper et al 2015). Transfer of human lactoferrin driven by the carp b-actin promoter to grass carp (Ctenopharyngodon idellus) delayed the onset of clinical signs in grass carp fry injected with grass carp haemorrhagic virus (GCHV) (Zhong et al 2002).…”