Gene-edited pigs for agricultural and biomedical applications are typically generated using somatic cell nuclear transfer(SCNT).However, SCNT requires the use of monoclonal cells as donors, and the time-consuming and ...Gene-edited pigs for agricultural and biomedical applications are typically generated using somatic cell nuclear transfer(SCNT).However, SCNT requires the use of monoclonal cells as donors, and the time-consuming and laborious monoclonal selection process limits the production of large populations of gene-edited animals. Here, we developed a rapid and efficient method named RE-DSRNP(reporter RNA enriched dual-sg RNA/CRISPR-Cas9 ribonucleoproteins) for generating gene-edited donor cells. RE-DSRNP takes advantage of the precise and efficient editing features of dual-sg RNA and the high editing efficiency, low off-target effects, transgene-free nature, and low cytotoxic characteristics of reporter RNA enriched RNPs(CRISPR-Cas9ribonucleoproteins), thus eliminating the need for the selection of monoclonal cells and thereby greatly reducing the generation time of donor cells from 3–4 weeks to 1 week, while also reducing the extent of apoptosis and chromosomal aneuploidy of donor cells. We applied RE-DSRNP to produce cloned pigs bearing a deletion edit of the wild-type p53-induced phosphatase 1(WIP1)gene: among 32 weaned cloned pigs, 31(97%) carried WIP1 edits, and 15(47%) were homozygous for the designed fragment deletion, and no off-target event was detected. The WIP1 knockout(KO) pigs exhibited male reproductive disorders, illustrating the utility of RE-DSRNP for rapidly generating precisely edited animals for functional genomics and disease research. REDSRNP's strong editing performance in a large animal and its marked reduction in the required time for producing SCNT donor cells support its application prospects for rapidly generating populations of transgene-free cloned animals.展开更多
There are still controversies about the roles of microRNA-26a（miR-26a）in human malignancies,as it is a tumor suppressor in breast cancer,gastric cancer,and hepatocellular carcinoma,but is an oncogene in glioma and c...There are still controversies about the roles of microRNA-26a（miR-26a）in human malignancies,as it is a tumor suppressor in breast cancer,gastric cancer,and hepatocellular carcinoma,but is an oncogene in glioma and cholangiocarcinoma.Until now,the function of miR-26a in osteosarcoma remains largely elusive.Here,we found that miR-26a was downregualted in osteosarcoma tissues.Using in vitro and in vivo assays,we confirmed that miR-26a could inhibit the abilities of in vitro proliferation and suppress in vivo tumor growth in mouse model.Furthermore,we identified insulin-like growth factor 1（IGF-1）as a novel and direct target of miR-26a and revealed that miR-26a exerted its tumor-suppressor function,at least in part,by inhibiting IGF-1expression.These findings contribute to our understanding of the functions of miR-26a in osteosarcoma.展开更多
The average number of available oocytes recovered per ovary collected during the breeding season in dairy goats was 5.5 (1815/330). 66.17% (1201/1815) of oocytes extruded the first polar body after maturation in vitro...The average number of available oocytes recovered per ovary collected during the breeding season in dairy goats was 5.5 (1815/330). 66.17% (1201/1815) of oocytes extruded the first polar body after maturation in vitro for 20 h. 75.44% (906/1201) of matured oocytes with membrane evagination around the MII chromosomes were enucleated. Ear skin fibroblast cells were derived from an adult female Jining Grey goat (C. hircus). The cells were cryopreserved in liquid nitrogen after passage 2. Thawed cells were further cultured for 3-6 passages and were subjected to serum starvation by 0.5% FBS for 2-10 d, then used as donor cells for nuclear transfer. 98.12% (889/906) of the enucleated oocytes were reconstructed by intracytoplasmic injection of karyoplast. The reconstructed embryos were activated by 5μmol/L ionomycin for 4.5 min and further activated by culturing with 6-dimethylaminopurine (6-DMAP) for 3 h. After 36 h of culture in mCR1aaBF, 76.69% (645/841) of the cloned embryos cleaved. There were no significant differences in development in vitro between the cloned embryos derived from donor cells pre-cooled at 4℃ for 24 h and nonprecooled donor cells. The cleavage rates, 4-cell development, and blastocyst development of reconstructed embryos were 72.48% (79/109), 53.16% (42/79), and 19.05% (8/42) in precooled group; 68.5% (211/308), 59.72% (126/211), and 17.46% (22/126) in nonprecooled group, respectively. Eighteen cloned 4-cell embryos derived from precooled donor cells were transferred and one cloned kid was born. Eighty-four cloned 4-cell embryos derived from nonprecooled donor cells were transferred and no offspring were produced. Of 18 cloned morale from nonprecooled donor cells transferred, one kid was born. The results of microsatellite DNA analyses indicated that the two cloned kids were from the same donor fibroblast cell line derived from an adult goat ear skin.展开更多
Dear Editors,Sexual dimorphism is the systematic difference in size,shape,color,physiology,and behavior,between male and female individuals of the same species（Mei and Gui,2015）.Some studies have indicated that the ...Dear Editors,Sexual dimorphism is the systematic difference in size,shape,color,physiology,and behavior,between male and female individuals of the same species（Mei and Gui,2015）.Some studies have indicated that the traits of sexual dimorphism in vertebrates are the consequences of sex-biased gene expression and are controlled by multiple critical genes during growth and development（Williams and Carroll,2009）.However,the exact molecular mechanism underlying sexual dimorphism remains unclear.展开更多
Dear Editor,Coronaviruses are enveloped positive-strand RNA viruses with 27–33 kb genomes.These viruses are classified into four genera,namely Alphacoronavirus,Betacoronavirus,Gammacoronavirus,
Short-term cultured cumulus cell lines (1-5BCC) derived from 5 individual cows were used in nuclear transfer (NT) and 1188 enucleated bovine oocytes matured in vitro were used as nuclear recipients. A total of 931 (78...Short-term cultured cumulus cell lines (1-5BCC) derived from 5 individual cows were used in nuclear transfer (NT) and 1188 enucleated bovine oocytes matured in vitro were used as nuclear recipients. A total of 931 (78.4%) cloned embryos were reconstructed, of which 763 (82%) cleaved, 627 (67.3%) developed to 8-cell stage, and 275 (29.5%) reached blastocyst stage. The average cell number of blastocysts was 124±24.5 (n=20). In this study, the effects of donor cell sources, serum starvation of donor cells, time interval from fusion to activation (IFA) were also tested on cloning efficiency. These results showed that blastocyst rates of embryos reconstructed from 5 different individuals cells were significantly different among them (14.1%, 45.2%, 27.3%, 34.3%, vs 1.5%, P<0.05); that serum starvation of donor cells had no effect on blastocyst development rate of NT embryos (47.1% vs 44.4%, P>0.05); and that blastocyst rate (20.3%) of the group with fusion/activation interval of 2-3 h, was significantly lower than that of the 3-6 h groups (31.0%), while not significantly different among 3-4 h (P < 0.05), 4-5 h, and 5-6 h groups (P ≥ 0.05). Sixty-three thawed NT blastocysts were transferred to 31 recipient cows, of which 4 pregnancies were established and two cloned calves were given birth. These results indicate that serum starvation of cumulus cells is not a key factor for successful bovine cloning, while IFA treatment and sources of donor cells have effects on cloning efficiency.展开更多
Somatic nuclear transfer technology has become increasingly promising in biomedicine and agriculture.Whereas the approach remains inefficient and underlying mechanisms remain ambiguous.Although cloned embryos have sim...Somatic nuclear transfer technology has become increasingly promising in biomedicine and agriculture.Whereas the approach remains inefficient and underlying mechanisms remain ambiguous.Although cloned embryos have similar in vitro developmental capacity as in vitro fertilized（IVF） embryos before implantation,they appeared to have much lower full-term developmental efficiency in pig and cattle,and thus it would be reasonable to postulate that profound distinction at the molecular level should exist between them.Herein,RNA sequencing technique was used to screen differentially expressed genes in cloned and IVF blastocysts,and in total 628 differentially expressed transcripts were obtained,among which,280 transcripts are up-regulated and 348 transcripts are down-regulated in cloned blastocysts.Moreover,one statistically significant pathway associated with endoplasmic reticulum（ER） protein processing was enriched,and some ER-stress markers such as ATF4,ATF6,PDIA3,HSPA1 B,HSP40 and HSP90 between cloned and IVF blastocysts were suggested.Additionally,some developmentally important genes such as lipid metabolism related genes（MGLL,DDHD2 and FADS2） and epigenetic modification genes（DNMT1,KDM5 C and MBD3L5） were found differentially expressed between cloned and IVF embryos.展开更多
Complementation of mutation in E. coli has been successful in cloning specific eukaryotic genes of yeast and animal cells for use in transformation. As a metabolic pathway of housekeeping protein, the synthesis of pro...Complementation of mutation in E. coli has been successful in cloning specific eukaryotic genes of yeast and animal cells for use in transformation. As a metabolic pathway of housekeeping protein, the synthesis of proline and its regulation might be conservative in prokaryotic and eukaryotic cells. With this in mind, we had cloned pro^+ genes from mouse and frog using this method in 1980.展开更多
Dear Editor,The 2002–2003 severe acute respiratory syndrome coronavirus（SARS-CoV）（Drosten et al.,2003）caused human pandemics that began in China and spread globally.Subsequently,
We introduce a new method for separation/enrichment of the low-content cellular protein in high mo-lecular weight on the basis of molecular imprinting. The template protein, bacterial cloned immu-noglobulin binding pr...We introduce a new method for separation/enrichment of the low-content cellular protein in high mo-lecular weight on the basis of molecular imprinting. The template protein, bacterial cloned immu-noglobulin binding protein (BiP), was selectively assembled with assistant recognition polymer chains (ARPCs) from their library, which consists of numerous limited length polymer chains with randomly distributed recognition and immobilizing sites. The assemblies of proteins and ARPCs were adsorbed by porous polymeric beads and immobilized by cross-linking polymerization. After the template was removed, the synthesized imprinted polymer was used to adsorb authentic BiP from endoplasmic re-ticulum (ER) extract, and its proportional content was enriched 45 times. It is the first time that the low-content cellular natural protein, whose molecular weight reaches 78 kDa, is enriched by molecular imprinting.展开更多
Somatic cell nuclear transfer(SCNT)-derived piglets have signi?cantly higher stillbirth rate and postnatal mortality rate than arti?cial insemination(AI)-generated piglets. The question whether the low survival rate o...Somatic cell nuclear transfer(SCNT)-derived piglets have signi?cantly higher stillbirth rate and postnatal mortality rate than arti?cial insemination(AI)-generated piglets. The question whether the low survival rate of SCNT piglets was related to birth weight, umbilical cord or placenta development was investigated. In this study,stillbirth rate, neonatal death rate, birth weight, umbilical cord status, placental parameters and placental gene expression patterns were compared between SCNT and AI piglets. Results showed that mortality rates at birth and during the neonatal stage of SCNT piglets were signi?-cantly higher than those of AI piglets. The incidence of abnormal umbilical cord in SCNT and SCNT-liveborn(SCNT-LB) piglets was signi?cantly higher than in AI and AI-liveborn(AI-LB) piglets. Birth weight, placental weight, placental surface area and placental ef?ciency in SCNT and SCNT-LB piglets were signi?cantly lower than those of AI and AI-LB piglets. Placental expression pro?les of imprinting, angiopoiesis and nutrient transportrelated genes were defective in SCNT-LB piglets compared with those in AI-LB piglets. Thus, the low survival rate of SCNT piglets may be associated with abnormal umbilical cord and placenta development. These characteristics may have resulted from aberrant expression of angiogenesis, nutrient transport, and imprinting-related genes in the placentas.展开更多
Objective To test what length is needed for polyamine binding both intrinsic gate and pore docking site to block the cloned strong inwardly rectifying channel (Kir2.1 channel).Methods The effect of alkylamine analogue...Objective To test what length is needed for polyamine binding both intrinsic gate and pore docking site to block the cloned strong inwardly rectifying channel (Kir2.1 channel).Methods The effect of alkylamine analogues(DA5,DA8,DA10 and DA12) and the competitive interaction of polyamine toxin,philanthotoxin(PhTx),on expressed Kir 2.1channel in Xenopus oocytes were examined by using giant excised inside out patch clamp technique.Results The results showed that along with the increase of the length of DAs ,the value of Kd decreased and the high affinity binding increased gradually.However,PhTx had the strongest effect on interfering the DA10 binding between intrinsic gate and pore docking site and had a less effect on DA12.Conclusion DA10 may be the right length for polyamine to block the channel.And maybe there is a hydrophobic interaction between DA12 and C terminal domain of this channel, which then stabilize the DA12 binding between these two points and decrease the effect of PhTx on DA12.展开更多
To explore the structural characteristic of ribosomal protein S29 (rpS29) gene of Ailuropoda melanoleuca (giant panda), primers were designed based on the known nucleotide sequence of rpS29 genes to clone the cDNA and...To explore the structural characteristic of ribosomal protein S29 (rpS29) gene of Ailuropoda melanoleuca (giant panda), primers were designed based on the known nucleotide sequence of rpS29 genes to clone the cDNA and genomic sequences of this gene from giant panda by RT-PCR and PCR strategy respectively, and then the cloned cDNA and genomic sequences were sequenced and analyzed preliminarily. The results indicated that the cDNA fragment of the rpS29 from the giant panda is 205bp in size in length, containing an open reading frame (ORF) of 171bp, encoding 56 amino acids. The length of the genomic sequence is 1 598 bp, with three exons and two introns. The coding sequence shows a high degree of homology to those of Homo sapiens, Bos taurus, Rattus norvegicus and Mus musculus with 75%, 94.74%, 89.47% and 88.89% respectively, While all the homologies for amino acid sequences are high up to 100%. Primary structure analysis revealed that the molecular weight of the putative rpS29 protein is 6.68 KD, with a theoretical pI of 10.63. Based on topology prediction, there are three distinct types of functional sites in the rpS29 protein of giant panda.展开更多
The complete gene coding for human neutrophilactivating protein-1/interleukin-8 was synthesized using a semi-chemical semi-enzymatic method. The synthetic gene was then overexpressed in Escherichia coli under the temp...The complete gene coding for human neutrophilactivating protein-1/interleukin-8 was synthesized using a semi-chemical semi-enzymatic method. The synthetic gene was then overexpressed in Escherichia coli under the temperature-regulated control of the P_RP_L tandem promoters. As determined by SDS-PAGE and densitometry, the overexpressed protein comprised up to 18.5% and 10.9% of the total soluble protein in E. coli cells grown in shake flasks and in batch fermentation, respectively. The recombinant NAP-1/IL-8 was then purified to>95% homogeneity by gel filtration and cation exchange chromatography. The purified protein appeared as a single band on the SDS-PAGE gel and possessed potent chemotactic activity in the concentration of <10 ng/ml, as assayed by the agarose plate method. An early skin reactivity was also observed when the pure NAP-1/IL-8 was injected subcutaneously into the rabbits. The N-terminal 36 amino acid sequence of the recombinant NAP1/IL-8 was determined using the Edman method and was shown to be identical to that of the ntive protein.展开更多
In order to understand molecular characterization of Citrus tatter leaf virus（CTLV） isolated from China,full-length cDNAs of CTLV-MTH and CTLV-XHC from Citrus reticulata and Citrus sinensis were cloned and sequenced...In order to understand molecular characterization of Citrus tatter leaf virus（CTLV） isolated from China,full-length cDNAs of CTLV-MTH and CTLV-XHC from Citrus reticulata and Citrus sinensis were cloned and sequenced based on whole-genome amplification by RT-PCR.The complete nucleotide sequences of CTLV-MTH and CTLV-XHC were determined to be 6497 nucleotides in length and shared 79.9-91.0%and 78.8-98.0%nucleotide sequence identity,respectively,with other Apple stem grooving virus（ASGV） or CTLV strains available in GenBank.Unexpectedly,CTLV-MTH showed the highest nucleotide sequence identity（91%） with an apple isolate of ASGV,followed by 86.5%with ASGV-HH and 85.7%with ASGV-CHN.Furthermore,CTLV-MTH and three ASGV strains were grouped to a separate cluster in the phylogenetic tree,suggesting it has a closer relationship to ASGV than to CTLV.Therefore,it can be concluded roughly that CTLV may be not a distinct strains of ASGV.We proposed that Citrus tatter leaf virus should be renamed Apple stem grooving virus.展开更多
The large protein of the hepatitis B virus (HBV) envelope was subdivided intothe preSl region with 108-115 amino acids, the preS2 region with 55 amino acidsand the S region with 226 amino acids. It was believed that t...The large protein of the hepatitis B virus (HBV) envelope was subdivided intothe preSl region with 108-115 amino acids, the preS2 region with 55 amino acidsand the S region with 226 amino acids. It was believed that the preSl region cotainedthe hepatocyte attachment sites of the virus as well as multiple highly immunogenicT-cell and B-cell epitopes. The antibody response to the preSl peptide was also oneof the most important serological markers of HBV infection.展开更多
基金supported by the National Natural Science Foundation of China (32072690)the Major Scientific Research Tasks for Scientific and Technological Innovation Projects of the Chinese Academy of Agricultural Sciences (CAAS-ZDRW202006)+3 种基金the Science, Technology and Innovation Commission of Shenzhen Municipality (JCKYZDKY202009)the National Transgenic Breeding Project (2016ZX08010-004)the National Transgenic Breeding Project (2016ZX08006-001)the Agricultural Science and Technology Innovation Program (ASTIP-IAS05)。
文摘Gene-edited pigs for agricultural and biomedical applications are typically generated using somatic cell nuclear transfer(SCNT).However, SCNT requires the use of monoclonal cells as donors, and the time-consuming and laborious monoclonal selection process limits the production of large populations of gene-edited animals. Here, we developed a rapid and efficient method named RE-DSRNP(reporter RNA enriched dual-sg RNA/CRISPR-Cas9 ribonucleoproteins) for generating gene-edited donor cells. RE-DSRNP takes advantage of the precise and efficient editing features of dual-sg RNA and the high editing efficiency, low off-target effects, transgene-free nature, and low cytotoxic characteristics of reporter RNA enriched RNPs(CRISPR-Cas9ribonucleoproteins), thus eliminating the need for the selection of monoclonal cells and thereby greatly reducing the generation time of donor cells from 3–4 weeks to 1 week, while also reducing the extent of apoptosis and chromosomal aneuploidy of donor cells. We applied RE-DSRNP to produce cloned pigs bearing a deletion edit of the wild-type p53-induced phosphatase 1(WIP1)gene: among 32 weaned cloned pigs, 31(97%) carried WIP1 edits, and 15(47%) were homozygous for the designed fragment deletion, and no off-target event was detected. The WIP1 knockout(KO) pigs exhibited male reproductive disorders, illustrating the utility of RE-DSRNP for rapidly generating precisely edited animals for functional genomics and disease research. REDSRNP's strong editing performance in a large animal and its marked reduction in the required time for producing SCNT donor cells support its application prospects for rapidly generating populations of transgene-free cloned animals.
文摘There are still controversies about the roles of microRNA-26a（miR-26a）in human malignancies,as it is a tumor suppressor in breast cancer,gastric cancer,and hepatocellular carcinoma,but is an oncogene in glioma and cholangiocarcinoma.Until now,the function of miR-26a in osteosarcoma remains largely elusive.Here,we found that miR-26a was downregualted in osteosarcoma tissues.Using in vitro and in vivo assays,we confirmed that miR-26a could inhibit the abilities of in vitro proliferation and suppress in vivo tumor growth in mouse model.Furthermore,we identified insulin-like growth factor 1（IGF-1）as a novel and direct target of miR-26a and revealed that miR-26a exerted its tumor-suppressor function,at least in part,by inhibiting IGF-1expression.These findings contribute to our understanding of the functions of miR-26a in osteosarcoma.
基金This work was finished in Northwest Sci-tech University of Agriculture and Forestry. We thankProf. Chen Sumin, Chen Nanchun and Dr. Chai Yubo for microsatellite DNA analysis. And we thank Dr. Wang Xinzhuang, Liu Zelong for help of embryos transfer in g
文摘The average number of available oocytes recovered per ovary collected during the breeding season in dairy goats was 5.5 (1815/330). 66.17% (1201/1815) of oocytes extruded the first polar body after maturation in vitro for 20 h. 75.44% (906/1201) of matured oocytes with membrane evagination around the MII chromosomes were enucleated. Ear skin fibroblast cells were derived from an adult female Jining Grey goat (C. hircus). The cells were cryopreserved in liquid nitrogen after passage 2. Thawed cells were further cultured for 3-6 passages and were subjected to serum starvation by 0.5% FBS for 2-10 d, then used as donor cells for nuclear transfer. 98.12% (889/906) of the enucleated oocytes were reconstructed by intracytoplasmic injection of karyoplast. The reconstructed embryos were activated by 5μmol/L ionomycin for 4.5 min and further activated by culturing with 6-dimethylaminopurine (6-DMAP) for 3 h. After 36 h of culture in mCR1aaBF, 76.69% (645/841) of the cloned embryos cleaved. There were no significant differences in development in vitro between the cloned embryos derived from donor cells pre-cooled at 4℃ for 24 h and nonprecooled donor cells. The cleavage rates, 4-cell development, and blastocyst development of reconstructed embryos were 72.48% (79/109), 53.16% (42/79), and 19.05% (8/42) in precooled group; 68.5% (211/308), 59.72% (126/211), and 17.46% (22/126) in nonprecooled group, respectively. Eighteen cloned 4-cell embryos derived from precooled donor cells were transferred and one cloned kid was born. Eighty-four cloned 4-cell embryos derived from nonprecooled donor cells were transferred and no offspring were produced. Of 18 cloned morale from nonprecooled donor cells transferred, one kid was born. The results of microsatellite DNA analyses indicated that the two cloned kids were from the same donor fibroblast cell line derived from an adult goat ear skin.
基金supported by the State Key Laboratory of Freshwater Ecology and Biotechnology (2015FB03)the Fundamental Research Funds for the Central Universities (52204-12018, 2013PY068)
文摘Dear Editors,Sexual dimorphism is the systematic difference in size,shape,color,physiology,and behavior,between male and female individuals of the same species（Mei and Gui,2015）.Some studies have indicated that the traits of sexual dimorphism in vertebrates are the consequences of sex-biased gene expression and are controlled by multiple critical genes during growth and development（Williams and Carroll,2009）.However,the exact molecular mechanism underlying sexual dimorphism remains unclear.
基金supported by grants from the State Megaproject for Infectious Disease Research of China(2014ZX10004001-002,2013ZX10004101,2013ZX10004805-002)
文摘Dear Editor,Coronaviruses are enveloped positive-strand RNA viruses with 27–33 kb genomes.These viruses are classified into four genera,namely Alphacoronavirus,Betacoronavirus,Gammacoronavirus,
基金This work was supported by the National "863" Project in China and Beijing Municipal Natural Sciences Foundation.
文摘Short-term cultured cumulus cell lines (1-5BCC) derived from 5 individual cows were used in nuclear transfer (NT) and 1188 enucleated bovine oocytes matured in vitro were used as nuclear recipients. A total of 931 (78.4%) cloned embryos were reconstructed, of which 763 (82%) cleaved, 627 (67.3%) developed to 8-cell stage, and 275 (29.5%) reached blastocyst stage. The average cell number of blastocysts was 124±24.5 (n=20). In this study, the effects of donor cell sources, serum starvation of donor cells, time interval from fusion to activation (IFA) were also tested on cloning efficiency. These results showed that blastocyst rates of embryos reconstructed from 5 different individuals cells were significantly different among them (14.1%, 45.2%, 27.3%, 34.3%, vs 1.5%, P<0.05); that serum starvation of donor cells had no effect on blastocyst development rate of NT embryos (47.1% vs 44.4%, P>0.05); and that blastocyst rate (20.3%) of the group with fusion/activation interval of 2-3 h, was significantly lower than that of the 3-6 h groups (31.0%), while not significantly different among 3-4 h (P < 0.05), 4-5 h, and 5-6 h groups (P ≥ 0.05). Sixty-three thawed NT blastocysts were transferred to 31 recipient cows, of which 4 pregnancies were established and two cloned calves were given birth. These results indicate that serum starvation of cumulus cells is not a key factor for successful bovine cloning, while IFA treatment and sources of donor cells have effects on cloning efficiency.
基金supported by a grant from the NationalHigh-Technology Research and Development Program of China(2011AA100304)two grants from Guangdong Provincial Department of Science and Technology,China(2011A090700016 and 2011A020102003)
文摘Somatic nuclear transfer technology has become increasingly promising in biomedicine and agriculture.Whereas the approach remains inefficient and underlying mechanisms remain ambiguous.Although cloned embryos have similar in vitro developmental capacity as in vitro fertilized（IVF） embryos before implantation,they appeared to have much lower full-term developmental efficiency in pig and cattle,and thus it would be reasonable to postulate that profound distinction at the molecular level should exist between them.Herein,RNA sequencing technique was used to screen differentially expressed genes in cloned and IVF blastocysts,and in total 628 differentially expressed transcripts were obtained,among which,280 transcripts are up-regulated and 348 transcripts are down-regulated in cloned blastocysts.Moreover,one statistically significant pathway associated with endoplasmic reticulum（ER） protein processing was enriched,and some ER-stress markers such as ATF4,ATF6,PDIA3,HSPA1 B,HSP40 and HSP90 between cloned and IVF blastocysts were suggested.Additionally,some developmentally important genes such as lipid metabolism related genes（MGLL,DDHD2 and FADS2） and epigenetic modification genes（DNMT1,KDM5 C and MBD3L5） were found differentially expressed between cloned and IVF embryos.
文摘Complementation of mutation in E. coli has been successful in cloning specific eukaryotic genes of yeast and animal cells for use in transformation. As a metabolic pathway of housekeeping protein, the synthesis of proline and its regulation might be conservative in prokaryotic and eukaryotic cells. With this in mind, we had cloned pro^+ genes from mouse and frog using this method in 1980.
基金funded by the National Natural Science Foundation of China (81290341)China Mega-Project for Infectious Disease (2014ZX10004001-003) from the Minister of Science and Technology of the People’s Republic of ChinaUSNIAID (R01AI110964)
文摘Dear Editor,The 2002–2003 severe acute respiratory syndrome coronavirus（SARS-CoV）（Drosten et al.,2003）caused human pandemics that began in China and spread globally.Subsequently,
基金Supported by the National Natural Science Foundation of China (Grant No. 20674040)
文摘We introduce a new method for separation/enrichment of the low-content cellular protein in high mo-lecular weight on the basis of molecular imprinting. The template protein, bacterial cloned immu-noglobulin binding protein (BiP), was selectively assembled with assistant recognition polymer chains (ARPCs) from their library, which consists of numerous limited length polymer chains with randomly distributed recognition and immobilizing sites. The assemblies of proteins and ARPCs were adsorbed by porous polymeric beads and immobilized by cross-linking polymerization. After the template was removed, the synthesized imprinted polymer was used to adsorb authentic BiP from endoplasmic re-ticulum (ER) extract, and its proportional content was enriched 45 times. It is the first time that the low-content cellular natural protein, whose molecular weight reaches 78 kDa, is enriched by molecular imprinting.
基金supported by two grants received from the Department of Science and Technology of Guangdong Province, China (2016B020233006 and 2016A020210074)
文摘Somatic cell nuclear transfer(SCNT)-derived piglets have signi?cantly higher stillbirth rate and postnatal mortality rate than arti?cial insemination(AI)-generated piglets. The question whether the low survival rate of SCNT piglets was related to birth weight, umbilical cord or placenta development was investigated. In this study,stillbirth rate, neonatal death rate, birth weight, umbilical cord status, placental parameters and placental gene expression patterns were compared between SCNT and AI piglets. Results showed that mortality rates at birth and during the neonatal stage of SCNT piglets were signi?-cantly higher than those of AI piglets. The incidence of abnormal umbilical cord in SCNT and SCNT-liveborn(SCNT-LB) piglets was signi?cantly higher than in AI and AI-liveborn(AI-LB) piglets. Birth weight, placental weight, placental surface area and placental ef?ciency in SCNT and SCNT-LB piglets were signi?cantly lower than those of AI and AI-LB piglets. Placental expression pro?les of imprinting, angiopoiesis and nutrient transportrelated genes were defective in SCNT-LB piglets compared with those in AI-LB piglets. Thus, the low survival rate of SCNT piglets may be associated with abnormal umbilical cord and placenta development. These characteristics may have resulted from aberrant expression of angiogenesis, nutrient transport, and imprinting-related genes in the placentas.
基金Present address:Departm ent of CardiologyFirst Hosptial of Xi'an Jiaotong University+1 种基金Xi'an 710 0 6 China
文摘Objective To test what length is needed for polyamine binding both intrinsic gate and pore docking site to block the cloned strong inwardly rectifying channel (Kir2.1 channel).Methods The effect of alkylamine analogues(DA5,DA8,DA10 and DA12) and the competitive interaction of polyamine toxin,philanthotoxin(PhTx),on expressed Kir 2.1channel in Xenopus oocytes were examined by using giant excised inside out patch clamp technique.Results The results showed that along with the increase of the length of DAs ,the value of Kd decreased and the high affinity binding increased gradually.However,PhTx had the strongest effect on interfering the DA10 binding between intrinsic gate and pore docking site and had a less effect on DA12.Conclusion DA10 may be the right length for polyamine to block the channel.And maybe there is a hydrophobic interaction between DA12 and C terminal domain of this channel, which then stabilize the DA12 binding between these two points and decrease the effect of PhTx on DA12.
基金financially supported by Application Foundation Project in Sichuan Province(2009JY0061)Youth Fund Project of Educational Committee of Sichuan province(09ZB088)
文摘To explore the structural characteristic of ribosomal protein S29 (rpS29) gene of Ailuropoda melanoleuca (giant panda), primers were designed based on the known nucleotide sequence of rpS29 genes to clone the cDNA and genomic sequences of this gene from giant panda by RT-PCR and PCR strategy respectively, and then the cloned cDNA and genomic sequences were sequenced and analyzed preliminarily. The results indicated that the cDNA fragment of the rpS29 from the giant panda is 205bp in size in length, containing an open reading frame (ORF) of 171bp, encoding 56 amino acids. The length of the genomic sequence is 1 598 bp, with three exons and two introns. The coding sequence shows a high degree of homology to those of Homo sapiens, Bos taurus, Rattus norvegicus and Mus musculus with 75%, 94.74%, 89.47% and 88.89% respectively, While all the homologies for amino acid sequences are high up to 100%. Primary structure analysis revealed that the molecular weight of the putative rpS29 protein is 6.68 KD, with a theoretical pI of 10.63. Based on topology prediction, there are three distinct types of functional sites in the rpS29 protein of giant panda.
文摘The complete gene coding for human neutrophilactivating protein-1/interleukin-8 was synthesized using a semi-chemical semi-enzymatic method. The synthetic gene was then overexpressed in Escherichia coli under the temperature-regulated control of the P_RP_L tandem promoters. As determined by SDS-PAGE and densitometry, the overexpressed protein comprised up to 18.5% and 10.9% of the total soluble protein in E. coli cells grown in shake flasks and in batch fermentation, respectively. The recombinant NAP-1/IL-8 was then purified to>95% homogeneity by gel filtration and cation exchange chromatography. The purified protein appeared as a single band on the SDS-PAGE gel and possessed potent chemotactic activity in the concentration of <10 ng/ml, as assayed by the agarose plate method. An early skin reactivity was also observed when the pure NAP-1/IL-8 was injected subcutaneously into the rabbits. The N-terminal 36 amino acid sequence of the recombinant NAP1/IL-8 was determined using the Edman method and was shown to be identical to that of the ntive protein.
基金supported by the Special Fund for Agro-Scientific Research in the Public Interest of China(201203076)the 111 Project,China(B12006)+1 种基金the Fundamental Research Funds for the Central Universities,China(XDJK2013C161,SWU113097)the Basic and Frontier Research Programs of Chongqing,China(CSTC2014jcyjA80033)
文摘In order to understand molecular characterization of Citrus tatter leaf virus（CTLV） isolated from China,full-length cDNAs of CTLV-MTH and CTLV-XHC from Citrus reticulata and Citrus sinensis were cloned and sequenced based on whole-genome amplification by RT-PCR.The complete nucleotide sequences of CTLV-MTH and CTLV-XHC were determined to be 6497 nucleotides in length and shared 79.9-91.0%and 78.8-98.0%nucleotide sequence identity,respectively,with other Apple stem grooving virus（ASGV） or CTLV strains available in GenBank.Unexpectedly,CTLV-MTH showed the highest nucleotide sequence identity（91%） with an apple isolate of ASGV,followed by 86.5%with ASGV-HH and 85.7%with ASGV-CHN.Furthermore,CTLV-MTH and three ASGV strains were grouped to a separate cluster in the phylogenetic tree,suggesting it has a closer relationship to ASGV than to CTLV.Therefore,it can be concluded roughly that CTLV may be not a distinct strains of ASGV.We proposed that Citrus tatter leaf virus should be renamed Apple stem grooving virus.
文摘The large protein of the hepatitis B virus (HBV) envelope was subdivided intothe preSl region with 108-115 amino acids, the preS2 region with 55 amino acidsand the S region with 226 amino acids. It was believed that the preSl region cotainedthe hepatocyte attachment sites of the virus as well as multiple highly immunogenicT-cell and B-cell epitopes. The antibody response to the preSl peptide was also oneof the most important serological markers of HBV infection.
