Efficiency of donor cell preparation and recipient oocyte source for production of transgenic cloned dairy goats harboring human lactoferrin

Wan, Yongjie; Zhou, Zhengrong; Jia, Ruoxin; Li, Meng; Song, Hui; Wang, Ziyu; Zhang, Guomin; You, Jia-Huai; Wang, Zhen

doi:10.1016/j.theriogenology.2012.03.004

Cited by 21 publications

(18 citation statements)

References 32 publications

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“…Furthermore, isolating fibroblast cells from two-month-old goats is easier and non-invasiveness to the animals. In our lab, seven lived transgenic cloned goat doeling has been obtained using transgenic fibroblast cells from three-month-old goats [9]. In another study in our lab using transgenic fibroblast cells from two-month-old goats, 47% pregnant percentage has been attained (unpublished data).…”

Section: Discussionmentioning

confidence: 89%

“…Many previous attempts have been intended to produce hLF for structure function studies [7], [8], but the previous methods were not suitable for very large-scale production. Transgenic goat mammary gland is an alternative way of producing this protein, because of large milk yield and precise posttranslational modifications [9]. Thus, we decided to investigate the effective way of producing hLF transgenic cloned dairy goats.…”

Section: Introductionmentioning

confidence: 99%

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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

Wan

Sun

et al. 2013

PLoS ONE

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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.

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Section: Discussionmentioning

confidence: 89%

Section: Introductionmentioning

confidence: 99%

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

Wan

Sun

et al. 2013

PLoS ONE

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“…Producing livestock by SCNT combination with a transgenic approach often requires genetic modification of donor cells. The cell line of the same origin was significantly different in the development rate of the NT embryo [27, 28]. In the present study, there was a significant difference in the cloning efficiency in different transgenic cell lines when used for nuclear transfer ( P < 0.05).…”

Section: Discussionmentioning

confidence: 57%

Expression of Recombinant Human Alpha-Lactalbumin in the Milk of Transgenic Goats Using a Hybrid Pomoter/Enhancer

Yuan

et al. 2014

Journal of Analytical Methods in Chemistry

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To improve nutrient content of goat milk, we describe the construction of a vector (pBLAC) containing a hybrid goat β-lactoglobulin (BLG) promoter/cytomegalovirus (CMV) enhancer. We also describe the generation of transgenic goats expressing rhLA by somatic cell nuclear transfer (SCNT). Of 334 one-cell stage embryos derived from three transgenic cell lines and 99 embryos derived from non-transgenic (NT) cells surgically transferred to the oviducts of 37 recipients, two recipients delivered two kids (2%) from the non-transfected line and five recipients delivered six kids (1.8%) from transgenic cell lines, three of which died within 2 days. Compared to the NT donor cells, transfection of donor cells does not negatively affect the development of nuclear transfer embryos into viable transgenic offspring. However, the clone efficiency in cell line number 1 was lower than that in numbers 2 and 3, and in the NT lines (0.9% versus 1.9% 2.4% and 2%; P < 0.05). Two transgenic cloned goats expressed rhLA in the milk at 0.1–0.9 mg/mL. The mammary gland-specific expression vector pBLAC with hybrid BLG/CMV can drive the hLA gene to express in vitro and in vivo. These data establish the basis for use of a hybrid promoter/enhancer strategy to produce rhLA transgenic goats.

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“…Deceased human lactoferrin (hLF) transgenic cloned goats were collected from previous studies that produced the goats using SCNT (Wan et al, 2012). Briefly, goat fibroblast cells from the ears of three-month-old goats were used to produce donor somatic cells harboring hLF for SCNT.…”

Section: Production Of Transgenic Goats and Sample Collectionmentioning

confidence: 99%

“…Briefly, goat fibroblast cells from the ears of three-month-old goats were used to produce donor somatic cells harboring hLF for SCNT. Culture and passage of donor cells, oocyte collection and enucleation, nuclear transfer, oocyte activation, embryo culture, and embryo transfer were carried out in order (Wan et al, 2012). Normal goats of the same age, produced using conventional reproduction, were used as controls.…”

Section: Production Of Transgenic Goats and Sample Collectionmentioning

confidence: 99%

Analysis of imprinted messenger RNA expression in deceased transgenic cloned goats

et al. 2016

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Efficiency of donor cell preparation and recipient oocyte source for production of transgenic cloned dairy goats harboring human lactoferrin

Cited by 21 publications

References 32 publications

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

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

Expression of Recombinant Human Alpha-Lactalbumin in the Milk of Transgenic Goats Using a Hybrid Pomoter/Enhancer

Analysis of imprinted messenger RNA expression in deceased transgenic cloned goats

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