共查询到20条相似文献,搜索用时 423 毫秒
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Eun Young KIM Dong Hwan SONG Min Jee PARK Hyo Young PARK Seung Eun LEE Hyun Yong CHOI Jeremiah Jiman MOON Young Hoon KIM Seong Ho MUN Chang Eon OH Moon Suck KO Dong Sun LEE Key Zung RIU Se Pill PARK 《The Journal of reproduction and development》2013,59(6):536-543
To preserve Jeju black cattle (JBC; endangered native Korean cattle), a pair of
cattle, namely a post-death cloned JBC bull and cow, were produced by somatic cell
nuclear transfer (SCNT) in a previous study. In the present study, we examined the
in vitro fertilization and reproductive potentials of these
post-death cloned animals. Sperm motility, in vitro fertilization
and developmental capacity were examined in a post-death cloned bull (Heuk Oll Dolee)
and an extinct nuclear donor bull (BK94-13). We assessed reproductive ability in
another post-death cloned cow (Heuk Woo Sunee) using cloned sperm for artificial
insemination (AI). There were no differences in sperm motility or developmental
potential of in vitro fertilized embryos between the post-death
cloned bull and its extinct nuclear donor bull; however, the embryo development ratio
was slightly higher in the cloned sperm group than in the nuclear donor sperm group.
After one attempt at AI, the post-death cloned JBC cow became pregnant, and gestation
proceeded normally until day 287. From this post-death cloned sire and dam, a JBC
male calf (Heuk Woo Dolee) was delivered naturally (weight, 25 kg). The genetic
paternity/maternity of the cloned JBC bull and cow with regard to their offspring was
confirmed using International Society for Animal Genetics standard microsatellite
markers. Presently, Heuk Woo Dolee is 5 months of age and growing normally. In
addition, there were no significant differences in blood chemistry among the
post-death cloned JBC bull, the cow, their offspring and cattle bred by AI. This is
the first report showing that a pair of cattle, namely, a post-death cloned JBC bull
and cow, had normal fertility. Therefore, SCNT can be used effectively to increase
the population of endangered JBC. 相似文献
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Masahito WATANABE Mirina KOBAYASHI Masaki NAGAYA Hitomi MATSUNARI Kazuaki NAKANO Miki MAEHARA Gota HAYASHIDA Shuko TAKAYANAGI Rieko SAKAI Kazuhiro UMEYAMA Nobuyuki WATANABE Masafumi ONODERA Hiroshi NAGASHIMA 《The Journal of reproduction and development》2015,61(3):169-177
Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36–37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear
donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum. 相似文献
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本研究以中国优良地方品种梅山猪为材料,采用胰酶消化法获得猪胎儿成纤维细胞,通过使用Y染色体SRY基因引物SRY-1、SRY-2进行PCR扩增,结果发现,雄性胎儿样本能扩增出250 bp的Y染色体特异性基因片段,而雌性胎儿样本未扩增出此条带;G1、G2、G3、G4和G5代的融合效率差异不显著(P>0.05),但G5代作为供体细胞核移植的重组胚胎卵裂率显著高于G1、G2、G3和G4代(P<0.05);使用G5代的胎儿成纤维细胞作为供核细胞,通过手术移植的方法,将重构胚胎移植到二元后备母猪体内,结果成功地获得了体细胞克隆梅山仔猪,并且仔猪全部为雄性胎儿,说明该性别鉴定方法不但操作简单,而且准确度高;经微卫星DNA 多态性鉴定,确定克隆猪来自供核细胞,与代孕母猪无亲缘关系。本研究将为中国优质猪品种改良、保种、性别控制及建立人类疾病模型等研究提供有效可行的方法,为批量生产克隆优秀种猪提供了坚实的理论基础。 相似文献
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Naomi NAKAGATA Toru TAKEO Kiyoko FUKUMOTO Yukie HARUGUCHI Tomoko KONDO Yumi TAKESHITA Yuko NAKAMUTA Tomoko UMENO Shuuji TSUCHIYAMA 《The Journal of reproduction and development》2014,60(2):168-171
Sperm cryopreservation has been widely adopted for maintenance of the genetically
engineered mouse (GEM). The cryopreserved sperm are being exchanged among many institutes
worldwide. However, the recipients are not always able to obtain high fertilization rates
with the frozen sperm shipped from senders. In this study, we cryopreserved mouse sperm
via various methods and performed in vitro
fertilization (IVF) in which the combination of methyl-beta-cyclodextrin for sperm
preincubation and reduced glutathione for insemination was used (the MBCD-GSH IVF). In
addition, frozen sperm sent from the Jackson Laboratory (USA) were thawed and used for IVF
in the same manner. The fertilization rates of both the sperm cryopreserved
via the methods applied in some countries and the cryopreserved GEM
sperm improved when used with the MBCD-GSH IVF method. Therefore, we strongly believe that
the MBCD-GSH IVF method brings about relatively high fertilization rates with any strain
of frozen mouse sperm. 相似文献
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Assessment of reproduction and growth performance of offspring derived from somatic cell cloned pigs
Zeping ZHAO Xinyu LU Biao LIU Yutian LI Hongbin WANG Zhonghua LIU 《Animal Science Journal》2012,83(9):639-643
Since cloned pig was successfully produced, a new opportunity for porcine breeding industry to conserve genetic resources has been opened. However, there has been no report to investigate whether both somatic cell nuclear transfer (SCNT) pigs and their offspring have the characteristics of the donor breed. In this study, we compared the reproductive and growth performance of American Large White boars cloned by SCNT with the donor boar, and analyzed the test parameters, including semen quality, re‐service rate, rate of parturition, and average daily gain. The results showed that these cloned boars and the donor boar had no significant differences in the tests (P > 0.05) and the growth performance of their offspring was similar to the naturally bred American Large White pigs. In summary, the reproductive and growth performance of cloned pigs are similar to the donor pig and within the normal range. This suggests that pigs cloned by SCNT have the potential to be used in reproduction and breeding. 相似文献
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Ok Jae Koo Seung-Kwon Ha Sol Ji Park Hee Jung Park Su Jin Kim Daekee Kwon Jung Taek Kang Joon Ho Moon Eun Jung Park Goo Jang Byeong Chun Lee 《Journal of veterinary science (Suw?n-si, Korea)》2015,16(2):241-244
Somatic cell nuclear transfer (SCNT) is a cost-effective technique for producing transgenic pigs. However, abnormalities in the cloned pigs might prevent use these animals for clinical applications or disease modeling. In the present study, we generated several cloned pigs. One of the pigs was found to have intrapancreatic ectopic splenic tissue during histopathology analysis although this animal was grossly normal and genetically identical to the other cloned pigs. Ectopic splenic tissue in the pancreas is very rare, especially in animals. To the best of our knowledge, this is the first such report for cloned pigs. 相似文献
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Mami OIKAWA Shogo MATOBA Kimiko INOUE Satoshi KAMIMURA Michiko HIROSE Narumi OGONUKI Hirosuke SHIURA Michihiko SUGIMOTO Kuniya ABE Fumitoshi ISHINO Atsuo OGURA 《The Journal of reproduction and development》2013,59(3):231-237
In mice, one of the major epigenetic errors associated with somatic cell nuclear
transfer (SCNT) is ectopic expression of Xist during the preimplantation
period in both sexes. We found that this aberrant Xist expression could
be impeded by deletion of Xist from the putative active X chromosome in
donor cells. In male clones, it was also found that prior injection of
Xist-specific siRNA could significantly improve the postimplantation
development of cloned embryos as a result of a significant repression of
Xist at the morula stage. In this study, we examined whether the same
knockdown strategy could work as well in female SCNT-derived embryos. Embryos were
reconstructed with cumulus cell nuclei and injected with Xist-specific
siRNA at 6–7 h after oocyte activation. RNA FISH analysis revealed that siRNA treatment
successfully repressed Xist RNA at the morula stage, as shown by the
significant decrease in the number of cloud-type Xist signals in the
blastomere nuclei. However, blastomeres with different sizes (from “pinpoint” to “cloud”)
and numbers of Xist RNA signals remained within single embryos. After
implantation, the dysregulated Xist expression was normalized
autonomously, as in male clones, to a state of monoallelic expression in both embryonic
and extraembryonic tissues. However, at term there was no significant improvement in the
survival of the siRNA-injected cloned embryos. Thus, siRNA injection was largely effective
in repressing the Xist overexpression in female cloned embryos but failed
to rescue them, probably because of an inability to mimic consistent monoallelic
Xist expression in these embryos. This could only be achieved in female
embryos by applying a gene knockout strategy rather than an siRNA approach. 相似文献
10.
Yuuki ISAJI Koki YOSHIDA Hiroshi IMAI Masayasu YAMADA 《The Journal of reproduction and development》2015,61(6):503-510
In mouse somatic cell nuclear transfer (SCNT), polyvinylpyrrolidone (PVP) is typically included in the
nuclear donor injection medium. However, the cytotoxicity of PVP, which is injected into the cytoplasm of
oocytes, has recently become a cause of concern. In the present study, we determined whether bovine serum
albumin deionized with an ion-exchange resin treatment (d-BSA) was applicable to the nuclear donor injection
medium in SCNT as an alternative to PVP. The results obtained showed that d-BSA introduced into the cytoplasm
of an enucleated oocyte together with a donor nucleus significantly enhanced the rate of in
vitro development of cloned embryos to the blastocyst stage compared with that of a conventional
nuclear injection with PVP in SCNT. We also defined the enhancing effects of d-BSA on the blastocyst formation
rate when d-BSA was injected into the cytoplasm of oocytes reconstructed using the fusion method with a
hemagglutinating virus of Japan envelope before oocyte activation. Furthermore, immunofluorescence experiments
revealed that the injected d-BSA increased the acetylation levels of histone H3 lysine 9 and histone H4 lysine
12 in cloned pronuclear (PN) and 2-cell embryos. The injection of d-BSA before oocyte activation also
increased the production of cloned mouse offspring. These results suggested that intracytoplasmic injection of
d-BSA into SCNT oocytes before oocyte activation was beneficial for enhancing the in vitro
and in vivo development of mouse cloned embryos through epigenetic modifications to nuclear
reprogramming. 相似文献
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Yan Jun HUAN Jiang ZHU Bing Teng XIE Jian Yu WANG Shi Chao LIU Yang ZHOU Qing Ran KONG Hong Bin HE Zhong Hua LIU 《The Journal of reproduction and development》2013,59(5):442-449
The efficiency of cloning by somatic cell nuclear transfer (SCNT) has remained low.
In most cloned embryos, epigenetic reprogramming is incomplete, and usually the
genome is hypermethylated. The DNA methylation inhibitor 5-aza-2’-deoxycytidine
(5-aza-dC) could improve the developmental competence of cow, pig, cat and human SCNT
embryos in previous studies. However, the parameters of 5-aza-dC treatment among
species are different, and whether 5-aza-dC could enhance the developmental
competence of porcine cloned embryos has still not been well studied. Therefore, in
this study, we treated porcine fetal fibroblasts (PFF) that then were used as donor
nuclei for nuclear transfer or fibroblast-derived reconstructed embryos with
5-aza-dC, and the concentration- and time-dependent effects of 5-aza-dC on porcine
cloned embryos were investigated by assessing pseudo-pronucleus formation,
developmental potential and pluripotent gene expression of these reconstructed
embryos. Our results showed that 5-aza-dC significantly reduced the DNA methylation
level in PFF (0 nM vs. 10 nM vs. 25 nM
vs. 50 nM, 58.70% vs. 37.37%
vs. 45.43% vs. 39.53%, P<0.05), but did not
improve the blastocyst rate of cloned embryos derived from these cells. Treating
cloned embryos with 25 nM 5-aza-dC for 24 h significantly enhanced the blastocyst
rate compared with that of the untreated group. Furthermore, treating cloned embryos,
but not donor cells, significantly promoted pseudo-pronucleus formation at 4 h post
activation (51% for cloned embryos treated, 34% for donor cells treated and 36% for
control, respectively, P<0.05) and enhanced the expression levels of pluripotent
genes (Oct4, Nanog and Sox2) up to
those of in vitro fertilized embryos during embryo development. In
conclusion, treating cloned embryos, but not donor cells, with 5-aza-dC enhanced the
developmental competence of porcine cloned embryos by promotion of pseudo-pronucleus
formation and improvement of pluripotent gene expression. 相似文献
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Miniature pigs share many similar characteristics such as anatomy, physiology and body size with humans and are expected to become important animal models for therapeutic cloning using embryonic stem cells (ESCs) derived by somatic cell nuclear transfer (SCNT). In the present study, we observed that miniature pig SCNT blastocysts possessed a lower total number of nuclei and a lower percentage of POU5F1‐positive cells than those possessed by in vitro fertilized (IVF) blastocysts. To overcome these problems, we evaluated the applicability of aggregating miniature pig SCNT embryos at the four‐cell stage. We showed that (i) aggregation of two or three miniature pig SCNT embryos at the four‐cell stage improves the total number of nuclei and the percentage of POU5F1‐positive cells in blastocysts, and (ii) IVF blastocysts with low cell numbers induced by the removal of two blastomeres at the four‐cell stage did not exhibit a decrease in the percentage of POU5F1‐positive cells. These results suggest that the aggregation of miniature pig SCNT embryos at the four‐cell stage can be a useful technique for improving the quality of miniature pig SCNT blastocysts and indicating that improvement in the percentage of POU5F1‐positive cells in aggregated SCNT embryos is not simply the consequence of increased cell numbers. 相似文献
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Xiao-ying He Li-bing Ma Xiao-ning He Wan-tong Si Yue-Mao Zheng 《Journal of veterinary science (Suw?n-si, Korea)》2016,17(2):145-152
Previous studies have established a bovine mammary gland epithelia cells in vitro model by the adenovirus-mediated telomerase (hTERT-bMGEs). The present study was conducted to confirm whether hTERT-bMGEs were effective target cells to improve the efficiency of transgenic expression and somatic cell nuclear transfer (SCNT). To accomplish this, a mammary-specific vector encoding human lysozyme and green fluorescent protein was used to verify the transgenic efficiency of hTERT-bMGEs, and untreated bovine mammary gland epithelial cells (bMGEs) were used as a control group. The results showed that the hTERT-bMGEs group had much higher transgenic efficiency and protein expression than the bMGEs group. Furthermore, the nontransgenic and transgenic hTERT-bMGEs were used as donor cells to evaluate the efficiency of SCNT. There were no significant differences in rates of cleavage or blastocysts or hatched blastocysts of cloned embryos from nontransgenic hTERT-bMGEs at passage 18 and 28 groups (82.8% vs. 81.9%, 28.6% vs. 24.8%, 58.6% vs. 55.3%, respectively) and the transgenic group (80.8%, 26.5% and 53.4%); however, they were significantly higher than the bMGEs group (71.2%, 12.8% and 14.8%), (p < 0.05). We confirmed that hTERT-bMGEs could serve as effective target cells for improving development of somatic cell cloned cattle embryos. 相似文献
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Yu-Ting Zhang Wang Yao Meng-Jia Chai Wen-Jing Liu Yan Liu Zhong-Hua Liu Xiao-Gang Weng 《Journal of veterinary science (Suw?n-si, Korea)》2022,23(2)
BackgroundSomatic cell nuclear transfer (SCNT) is used widely in cloning, stem cell research, and regenerative medicine. The type of donor cells is a key factor affecting the SCNT efficiency.ObjectivesThis study examined whether urine-derived somatic cells could be used as donors for SCNT in pigs.MethodsThe viability of cells isolated from urine was assessed using trypan blue and propidium iodide staining. The H3K9me3/H3K27me3 level of the cells was analyzed by immunofluorescence. The in vitro developmental ability of SCNT embryos was evaluated by the blastocyst rate and the expression levels of the core pluripotency factor. Blastocyst cell apoptosis was examined using a terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. The in vivo developmental ability of SCNT embryos was evaluated after embryo transfer.ResultsMost sow urine-derived cells were viable and could be cultured and propagated easily. On the other hand, most of the somatic cells isolated from the boar urine exhibited poor cellular activity. The in vitro development efficiency between the embryos produced by SCNT using porcine embryonic fibroblasts (PEFs) and urine-derived cells were similar. Moreover, The H3K9me3 in SCNT embryos produced from sow urine-derived cells and PEFs at the four-cell stage showed similar intensity. The levels of Oct4, Nanog, and Sox2 expression in blastocysts were similar in the two groups. Furthermore, there is a similar apoptotic level of cloned embryos produced by the two types of cells. Finally, the full-term development ability of the cloned embryos was evaluated, and the cloned fetuses from the urine-derived cells showed absorption.ConclusionsSow urine-derived cells could be used to produce SCNT embryos. 相似文献
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SJ Uhm MK Gupta ZC Das JH Kim C Park T Kim HT Lee 《Reproduction in domestic animals》2009,44(1):106-115
Retrovirus-mediated exogenous gene transfection of somatic cells is an efficient method to produce transgenic embryos by somatic cell nuclear transfer (SCNT). This study evaluated whether efficiency of transgenic embryos production, by SCNT using fibroblast cells transfected by retrovirus vector, is influenced by the introduced transgene and whether recloning could further improve its efficiency. Transgenic cloned embryos were produced by SCNT of porcine foetal fibroblast cells transfected by either LNβ-Z or LNβ-enhanced green fluorescent protein (EGFP) retrovirus vector and evaluated for their developmental ability in vitro . Blastomeres from four-cell stage porcine embryos, produced by SCNT of foetal fibroblast cells transfected with LNβ-EGFP retroviral vector, were subsequently recloned into enucleated metaphase II oocytes and evaluated for changes in chromatin configuration, in vitro embryo development and gene expression. Analysis of results showed that cleavage and blastocyst rates of porcine SCNT embryos, using LacZ (53.6 ± 6.4%; 12.0 ± 5.7%) or EGFP (57.5 ± 6.3%; 10.1 ± 4.1%) transfected fibroblasts, did not differ (p > 0.05) from those of non-transfected controls (60.9 ± 8.2%; 12.3 ± 4.0%). Recloning of blastomeres did not further improve the in vitro development rate. Interestingly, the nuclei of blastomere underwent slower remodelling process than somatic cell nuclei. Both cloned and recloned embryos showed 100% transgene expression and there were no evidence of mosaicism. In conclusion, our data shows that the efficiency of transgenic cloned embryos production by SCNT of somatic cells transfected with replication-defective retrovirus vector is not influenced by the transgene introduction into donor cells and recloning of four-cell stage blastomere could not further improve its efficiency. 相似文献
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Dong-Hoon Kim Jin-Gu No Mi-Kyung Choi Dong-Hyeon Yeom Dong-Kyo Kim Byoung-Chul Yang Jae Gyu Yoo Min Kyu Kim Hong-Tea Kim 《Journal of veterinary science (Suw?n-si, Korea)》2015,16(2):233-235
The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%) or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos. 相似文献
18.
L?rke B Astrup Mette V Nielsen Tine M Iburg Páll S Leifsson Henrik E Jensen Ole L Nielsen J?rgen S Agerholm 《Acta veterinaria Scandinavica》2013,55(1):76
Background
Sepsis caused by Staphylococcus aureus often leads to brain microabscesses in humans. Animal models of haematogenous brain abscesses would be useful to study this condition in detail. Recently, we developed a model of S. aureus sepsis in pigs and here we report that brain microabscesses develop in pigs with such induced S. aureus sepsis.Twelve pigs were divided into three groups. Nine pigs received an intravenous inoculation of S. aureus once at time 0 h (group 1) or twice at time 0 h and 12 h (groups 2 and 3). In each group the fourth pig served as control. The pigs were euthanized at time 12 h (Group 1), 24 h (Group 2) and 48 h (Group 3) after the first inoculation. The brains were collected and examined histopathologically.Results
All inoculated pigs developed sepsis and seven out of nine pigs developed brain microabscesses. The microabscesses contained S. aureus and were located in the prosencephalon and mesencephalon. Chorioditis and meningitis occurred from 12 h after inoculation.Conclusions
Pigs with experimental S. aureus sepsis often develop brain microabscesses. The porcine brain pathology mirrors the findings in human sepsis patients. We therefore suggest the pig as a useful animal model of the development of brain microabscesses caused by S. aureus sepsis. 相似文献19.
Cloned porcine embryos can maintain developmental ability after cryopreservation at the morula stage
Nakano K Matsunari H Nakayama N Ogawa B Kurome M Takahashi M Matsumoto M Murakami H Kaji Y Nagashima H 《The Journal of reproduction and development》2011,57(2):312-316
The aim of the present study was to clarify the overall efficiency of porcine somatic cell nuclear transfer (SCNT) by incorporating cryopreservation of the cloned embryos before transfer. The SCNT embryos reconstructed with preadipocytes and in vitro-matured (IVM) oocytes were cultured to harvest morula stage embryos; they were then subjected to delipation (removal of cytoplasmic lipid droplets) and vitrification. After warming and culture, the embryos developing to blastocysts were transferred to recipients to obtain cloned piglets. From 372 reconstructed embryos, 188 (50.5%) reached the morula stage and 117 (31.5%) developed to blastocysts after vitrification. Transfer of 98 (26.3%) morphologically normal blastocysts gave rise to 6 (1.6%) piglets, including 1 stillborn. The efficiency of the cloned piglet production was comparable with that obtained using SCNT embryos without cryopreservation (2.7%, 17/635). Here, we demonstrate that porcine somatic cell cloning can be performed without a significant reduction in efficiency even when the SCNT embryos are cryopreserved before transfer. 相似文献
20.
体细胞克隆猪的研究与展望 总被引:1,自引:1,他引:1
用体细胞核移植的方法生产的克隆猪诞生至今已近5年,虽然同胚胎细胞核移植相比,体细胞核移植技术难度大,成功率低,但近年来用体细胞核移植技术克隆猪有了更深入的研究,在体细胞核移植基本机理和关键技术上取得了一定的进展。综述了用核移植的方法生产体细胞克隆猪的技术关键和难点及应用情况,并对提高猪体细胞核移植效率的策略进行了分析。 相似文献