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.
Highlights
There are many ways available to produce transgenic animals, such as pronuclear injection and sperm-mediated gene transfer [1]
The results demonstrated that the methylation status of differential methylation regions (DMR) of H19 and Insulin-like growth factor 2 receptor (IGF2R) were different in lived and dead transgenic goats and this may be potentially used to assess the reprogramming status of transgenic cloned goats
Hyper-methylation of H19 and IGF2R genes was observed in CA goats, while DNA methylation in CL goats was not significantly different from NL goats
Summary
There are many ways available to produce transgenic animals, such as pronuclear injection and sperm-mediated gene transfer [1]. Somatic cell nuclear transfer (SCNT), which uses preselected genetically modified cells as donor nuclei, is recognized as a more efficient way to produce transgenic animals until now [2]. Dairy goats are preferable to other species to produce recombinant proteins in milk for the following reasons [5]. Dairy goats produce more milk than rabbits and mice. Transgenic goat mammary gland is an alternative way of producing this protein, because of large milk yield and precise posttranslational modifications [9]. We decided to investigate the effective way of producing hLF transgenic cloned dairy goats. Somatic cell nuclear transfer (SCNT) is a promising technique to produce transgenic cloned mammalian, including transgenic goats which may produce Human Lactoferrin (hLF). 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
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