Summary： Somatic cell nucleus transfer （SCNT） has been considered the most effective method for conserving endangered animals and expanding the quantity of adult animal models. Bama miniature pigs are genetically s...Summary： Somatic cell nucleus transfer （SCNT） has been considered the most effective method for conserving endangered animals and expanding the quantity of adult animal models. Bama miniature pigs are genetically stable and share similar biological features to humans. These pigs have been used to establish animal models for human diseases, and for many other applications. However, there is a pan- city of studies on the effect of ear fibroblasts derived from different age of adult Bama miniature pigs on nucleus transfer （NT）. The present study examined the NT efficiency of ear fibroblasts from fetal, new- born, 1-, 2-, 4-, 6-, 12-month-old miniature pigs by using trypan blue staining, flow cytometry and NT technique, etc., and the cell biological function and SCNT efficiency were compared between groups. The results showed that ear fibroblasts grew well after passage in each group. Spindle-shaped cells ini- tially predominated, and gradually declined with increase of culture time and replaced by polygonal cells. Irregular cell growth occurred in the 2-month-old group and the elder groups. The growth curves of the ear fibroblasts were ＂S-shaped＂ in different age groups. The cell proliferation of postnatal ear fi- broblasts, especially those from 2-, 4-, 6-, 12-month-old miniature pigs was significantly different from that of fetus ear fibroblasts （P〈0.05 or P〈0.01）. Two-month- and 4-month-old ear fibroblasts had a sig- nificantly higher proportion of G1 stage cells （85% to 91%） than those at 6 and 12 months （66% to 74%, P〈0.01）. The blastocyst rate of reconstructed embryos originating from newborn, 1-, 2-, 4-month-old donor pigs was 6.06% to 7.69% with no significant difference from that in fetus fibroblast group （8.06%）. It was concluded that 〈4-month-old adult Bama miniature pigs represent a better donor cell resource than elder pigs.展开更多
Transgenic pigs have been produced with the aim of further improving pigs in terms of economic and environmental traits, but these animals have not been allowed to enter the food chain. As an alternative approach to g...Transgenic pigs have been produced with the aim of further improving pigs in terms of economic and environmental traits, but these animals have not been allowed to enter the food chain. As an alternative approach to generating pigs with novel traits of economic importance that cannot be introduced by conventional breeding, we propose a strategy for combining in vitro mutagenesis of pig primary cells with N-ethyl-N-nitrosourea (ENU) and somatic-cell nuclear transfer (SCNT) technology. To explore the feasibility of this strategy, we treated pig primary fibroblast cells with ENU, estimated the per-base mutation frequency induced by the mutagen, clonally cultured about 4000 of the mutagenized cells, and screened them for mutation within the coding region of the melanocortin-4 receptor (MC4R) gene, a key gene in energy homeostasis. Through this screening, we obtained 14 cell clones, each harboring a heterozygous base change within the coding region for MC4R. Of the mutant cell clones, each of two contained a mutant allele encoding MC4R with greatly reduced receptor activity. By SCNT using these cell clones as donors, pigs harboring mutated MC4R alleles with reduced receptor activity can be produced. Our strategy for generating pigs with novel genetic traits likely will be more acceptable to the public than is the use of transgenic technology.展开更多
Homeobox A10(Hoxa10) gene is one of the most important candidate genes associated with the reproductive performance of humans and mice. Overexpression of Hoxa10 in mouse endometrium can increase litter size. Moreover,...Homeobox A10(Hoxa10) gene is one of the most important candidate genes associated with the reproductive performance of humans and mice. Overexpression of Hoxa10 in mouse endometrium can increase litter size. Moreover, Hoxa10 plays a key role in regulating the embryo implantation of sows. This study aimed to generate transgenic pigs using Hoxa10 via somatic cell nuclear transfer(SCNT). We established seven Hoxa10-transgenic cell lines, and two of the cell lines were selected as nuclear donors for the transfer. A total of 1 270 cloned embryos were generated and transferred to five surrogate mothers(Landrace×Yorkshire). Eight cloned male piglets were produced including one with cryptorchidism. Six transgenic piglets grew up healthy and produced 56 offspring. Finally, we obtained six transgenic male pigs and 26 transgenic positive offspring that can be used to further study the regulatory mechanism of Hoxa10 on the reproductive performance of pigs.展开更多
基金supported by the grants from the National Natural Science Foundation of China(No.31000546)National High-tech Research & Development Program of China(863 Program)(No.2012AA020603)+1 种基金National Science and Technology Major Project of China(No.2014zx08009-003-006)Rongchang Youth Foundation and Fundamental Research Funds of Southwest University(No.XDJK2012C097)
文摘Summary： Somatic cell nucleus transfer （SCNT） has been considered the most effective method for conserving endangered animals and expanding the quantity of adult animal models. Bama miniature pigs are genetically stable and share similar biological features to humans. These pigs have been used to establish animal models for human diseases, and for many other applications. However, there is a pan- city of studies on the effect of ear fibroblasts derived from different age of adult Bama miniature pigs on nucleus transfer （NT）. The present study examined the NT efficiency of ear fibroblasts from fetal, new- born, 1-, 2-, 4-, 6-, 12-month-old miniature pigs by using trypan blue staining, flow cytometry and NT technique, etc., and the cell biological function and SCNT efficiency were compared between groups. The results showed that ear fibroblasts grew well after passage in each group. Spindle-shaped cells ini- tially predominated, and gradually declined with increase of culture time and replaced by polygonal cells. Irregular cell growth occurred in the 2-month-old group and the elder groups. The growth curves of the ear fibroblasts were ＂S-shaped＂ in different age groups. The cell proliferation of postnatal ear fi- broblasts, especially those from 2-, 4-, 6-, 12-month-old miniature pigs was significantly different from that of fetus ear fibroblasts （P〈0.05 or P〈0.01）. Two-month- and 4-month-old ear fibroblasts had a sig- nificantly higher proportion of G1 stage cells （85% to 91%） than those at 6 and 12 months （66% to 74%, P〈0.01）. The blastocyst rate of reconstructed embryos originating from newborn, 1-, 2-, 4-month-old donor pigs was 6.06% to 7.69% with no significant difference from that in fetus fibroblast group （8.06%）. It was concluded that 〈4-month-old adult Bama miniature pigs represent a better donor cell resource than elder pigs.
文摘Transgenic pigs have been produced with the aim of further improving pigs in terms of economic and environmental traits, but these animals have not been allowed to enter the food chain. As an alternative approach to generating pigs with novel traits of economic importance that cannot be introduced by conventional breeding, we propose a strategy for combining in vitro mutagenesis of pig primary cells with N-ethyl-N-nitrosourea (ENU) and somatic-cell nuclear transfer (SCNT) technology. To explore the feasibility of this strategy, we treated pig primary fibroblast cells with ENU, estimated the per-base mutation frequency induced by the mutagen, clonally cultured about 4000 of the mutagenized cells, and screened them for mutation within the coding region of the melanocortin-4 receptor (MC4R) gene, a key gene in energy homeostasis. Through this screening, we obtained 14 cell clones, each harboring a heterozygous base change within the coding region for MC4R. Of the mutant cell clones, each of two contained a mutant allele encoding MC4R with greatly reduced receptor activity. By SCNT using these cell clones as donors, pigs harboring mutated MC4R alleles with reduced receptor activity can be produced. Our strategy for generating pigs with novel genetic traits likely will be more acceptable to the public than is the use of transgenic technology.
基金supported by the National Major Special Project on New Varieties Cultivation for Transgenic Organisms,China(2014ZX08006-005 and 2014ZX0800950B)
文摘Homeobox A10(Hoxa10) gene is one of the most important candidate genes associated with the reproductive performance of humans and mice. Overexpression of Hoxa10 in mouse endometrium can increase litter size. Moreover, Hoxa10 plays a key role in regulating the embryo implantation of sows. This study aimed to generate transgenic pigs using Hoxa10 via somatic cell nuclear transfer(SCNT). We established seven Hoxa10-transgenic cell lines, and two of the cell lines were selected as nuclear donors for the transfer. A total of 1 270 cloned embryos were generated and transferred to five surrogate mothers(Landrace×Yorkshire). Eight cloned male piglets were produced including one with cryptorchidism. Six transgenic piglets grew up healthy and produced 56 offspring. Finally, we obtained six transgenic male pigs and 26 transgenic positive offspring that can be used to further study the regulatory mechanism of Hoxa10 on the reproductive performance of pigs.
