Mutation of mevalonate kinase(MVK) is thought to account for most cases of hyperimmunoglobulinemia D syndrome(HIDS) with recurrent fever.However,its mechanism and the relationship between elevated serum immunoglobulin...Mutation of mevalonate kinase(MVK) is thought to account for most cases of hyperimmunoglobulinemia D syndrome(HIDS) with recurrent fever.However,its mechanism and the relationship between elevated serum immunoglobulin D(IgD) and the clinical features of HIDS are unclear.In this study,we generated by fusion PCR a vector to express high levels of chimeric secretory IgD(csIgD) specifically in the liver.We then generated seven founder lines of transgenic mice by co-microinjection,and verified them using genomic PCR and Southern blotting.We detected the expression of csIgD by reverse transcription PCR,quantitative PCR,western blotting,and enzyme-linked immunosorbent assays.We demonstrated that csIgD could be specifically and stably expressed in the liver.We used flow cytometry to show that overexpression of csIgD in the bone marrow and spleen cells had no effect on B cell development.Morphologic and anatomical observation of the transgenic mice revealed skin damage,hepatosplenomegaly,and nephromegaly in some transgenic mice;in these mice,pathological sections showed high levels of cell necrosis and protein-like sediments in the liver,spleen,and kidney.We demonstrated that the genomic insertion sites of the transgenes did not disrupt the MVK gene on mouse chromosome 5.This transgenic mouse will be useful to explore the pathogenesis of HIDS.展开更多
Six types of bovine somatic cell lines, including a granulosa cell line of Chinese red-breed yellow cattle (YGR), a granulosa cell line of Holstein cow (HGR), two skin fibroblast cell lines of two adult Holstein cows ...Six types of bovine somatic cell lines, including a granulosa cell line of Chinese red-breed yellow cattle (YGR), a granulosa cell line of Holstein cow (HGR), two skin fibroblast cell lines of two adult Holstein cows respectively (AFB1 and AFB2), a skin fibroblast cell line (FFB) and an oviduct epithelial cell line (FOV) of a Holstein fetus, were established. Somatic cell nu-clear transfer (SCNT) was carried out using these cells as nuclei donor, and a total of 12 healthy calves were cloned. The effects of different types of donor cells on developmental potential of bovine SCNT embryos were investigated. (i) There was no significant difference in development rates to the blastocyst stage for SCNT embryos from YGR and HGR (33.2% and 35.1%, respec-tively). Pregnancy rates of them were 33.3% and 30.2%, respectively; and birth rates were 16.7% and 11.6%, respectively. (ii) Development rates to the blastocyst stage for SCNT embryos from diffetent individuals (AFB1 and AFB2) differed significantly (27.9% and 39.4%, respectively, P <0.05). Pregnancy rates of them were 36.2% and 36.4%, respectively; and birth rates were 14.9 % and 27.3%, respectively. (iii) There was significant difference in development rates to the blastocyst stage for SCNT embryos from FFB and FOV of the same fetus (37.9% and 41.5%, respectively, P < 0.05). Pregnancy rates of them were 45.7% and 24.1%, respectively; and birth rates were 22.9 % and 10.3%, respectively. Finally, developmental potential of bovine SCNT embryos from all four types of somatic cells from Holstein cows (HGR, AFB, FFB and FOV) were compared. For in vitro development stage, development rates to the blastocyst stage for SCNT embryos from HGR, AFB, FFB and FOV were 35.1%A, 29.4%B, 37.9%A and 41.5%C, respectively (PABC<0.05); for in vivo development stage, pregnancy rates of them were 30.2%, 36.2%, 45.7% and 24.1%, respectively; and birth rates of them were 11.6%, 17.2%, 22.9% and 10.3% respec-tively.展开更多
In somatic cell nuclear transfer (SCNT) technologies,the donor cell’s nuclei need to be epigenetically reprogrammed for embryonic development. The incomplete reprogramming of donor cell nuclei has been implicated as ...In somatic cell nuclear transfer (SCNT) technologies,the donor cell’s nuclei need to be epigenetically reprogrammed for embryonic development. The incomplete reprogramming of donor cell nuclei has been implicated as a primary reason for the low efficiency of SCNT. DNA methylation is a major epige-netic modification of the genome that regulates crucial aspects of genome function,including estab-lishment of genomic imprinting. In order to make sure whether the DNA methylation reprogramming is efficient in SCNT animals,we analyzed the DNA methylation status of two imprinting genes,H19 and Xist,in lungs of deceased SCNT bovines that died within 48 h of birth using bisulfite sequencing analysis. Our findings demonstrated that cloned bovines showed significantly lower DNA methylation of H19 than controls (P<0.05),and three tested CpGs sites (1,2,3) exhibited unmethylation in one cloned bovine (9C3); however,Xist showed similar DNA methylation levels between clones and con-trols,and both showed hypermethylation (96.11% and 86.67%).展开更多
Cloning by somatic nuclear transfer is an inefficient process in which many of the cloned ani- mals died shortly after birth and displayed organ ab- normalities. In an effort to determine the possible genetic causes o...Cloning by somatic nuclear transfer is an inefficient process in which many of the cloned ani- mals died shortly after birth and displayed organ ab- normalities. In an effort to determine the possible genetic causes of neonatal death and organ abnor- malities, we have examined expression patterns of four genes that modified chromatin (DNMT1, PCAF,MeCP2 and EED) in six organs (heart, liver, spleen, lung, kidney and brain) of both neonatal death cloned bovines (n=9) and normal control calves produced by artificial insemination (AI) using real-time quantitative RT-PCR. The effect of the age of the fibroblast donor cell on the gene expression profiles was also investigated. Aberrant expressions of DNMT1 and PCAF were found in some studied tissues, but the expression of MeCP2 and EED had similar levels to those of the normal controls. The expression of DNMT1 showed a higher level in heart, liver and brain of both cloned bovines. A higher ex- pression level of PCAF was seen in heart and liver of both cloned bovines, but a lower level was seen only in spleen of adult fibroblast (AF) cell-derived clones. Our results suggest that aberrant expression in gene that modified chromatins were found in cloned bovine tissues of neonatal death. Because DNMT1 and PCAF play an important role in DNA methylation and histone acetylation on nuclear chromatin respectively, and normal expression of DNMT1 and PCAF is needed for precious reprogramming of donor nuclear, the aberrant transcription patterns of DNMT1 andPCAF in these clones may contribute to the defects of organs reported in neonatal death of clones.展开更多
Somatic cell clone technology is a viable ap- proach to preserving endangered livestock and wildlife ge- netic resources. In the present research, somatic cell nuclear transfer (SCNT) was performed using granulose cel...Somatic cell clone technology is a viable ap- proach to preserving endangered livestock and wildlife ge- netic resources. In the present research, somatic cell nuclear transfer (SCNT) was performed using granulose cells from the critical endangered Chinese red-cross yellow cattle as donor cells. A total of 211 oocytes were manipulated and 166 (79%) of them were successfully enucleated. 112 (67.4%) SCNT embryos were reconstructed, 94 (83%) of them cleaved, and 48 (43%) of them developed to blastocyst stage. SCNT blastocysts were transferred to 6 Holstein recipients, and 2 (33%) of them were found to be pregnant. One of them maintained to term and delivered a calf, whereas another aborted. Effect of different fusion buffer (mannitol vs. Zim- merman fusion buffer) and different activation methods (calcium ionophore+6-DMAP vs. cycloheximide+CB) on fu- sion rate and development of SCNT embryos were investi- gated. The results indicated that: (i) on condition of two DC pulses of 2.5 kV/cm for 10 μs each, fusion rates were higher in mannitol solution than in Zimmerman fusion buffer (71% vs. 61%, respectively, p<0.05), but the blastocysts rates did not differ between two treatments (36% vs. 39%, p>0.05 ); (ii) There was no significant difference in development rates to the blastocyst stage for SCNT embryos activated by calcium ionophore+6-DMAP or by cycloheximide+CB (42% vs. 46%, respectively, p>0.05). Microsatellite DNA analysis examining 28 loci confirmed that the cloned calf was genetically identi- cal to the donor Jinan red-cross yellow cattle and different from the recipient females. Growth and reproductive per- formance of cloned cow were evaluated, and there were no difference i cross-red n it between cloned and normal control Jinan yellow cattle. Furthermore, the cloned yellow cow has delivered a healthy yellow calf.展开更多
基金supported by the National Basic Research Program of China(Grant No.2010CB945300)
文摘Mutation of mevalonate kinase(MVK) is thought to account for most cases of hyperimmunoglobulinemia D syndrome(HIDS) with recurrent fever.However,its mechanism and the relationship between elevated serum immunoglobulin D(IgD) and the clinical features of HIDS are unclear.In this study,we generated by fusion PCR a vector to express high levels of chimeric secretory IgD(csIgD) specifically in the liver.We then generated seven founder lines of transgenic mice by co-microinjection,and verified them using genomic PCR and Southern blotting.We detected the expression of csIgD by reverse transcription PCR,quantitative PCR,western blotting,and enzyme-linked immunosorbent assays.We demonstrated that csIgD could be specifically and stably expressed in the liver.We used flow cytometry to show that overexpression of csIgD in the bone marrow and spleen cells had no effect on B cell development.Morphologic and anatomical observation of the transgenic mice revealed skin damage,hepatosplenomegaly,and nephromegaly in some transgenic mice;in these mice,pathological sections showed high levels of cell necrosis and protein-like sediments in the liver,spleen,and kidney.We demonstrated that the genomic insertion sites of the transgenes did not disrupt the MVK gene on mouse chromosome 5.This transgenic mouse will be useful to explore the pathogenesis of HIDS.
文摘Six types of bovine somatic cell lines, including a granulosa cell line of Chinese red-breed yellow cattle (YGR), a granulosa cell line of Holstein cow (HGR), two skin fibroblast cell lines of two adult Holstein cows respectively (AFB1 and AFB2), a skin fibroblast cell line (FFB) and an oviduct epithelial cell line (FOV) of a Holstein fetus, were established. Somatic cell nu-clear transfer (SCNT) was carried out using these cells as nuclei donor, and a total of 12 healthy calves were cloned. The effects of different types of donor cells on developmental potential of bovine SCNT embryos were investigated. (i) There was no significant difference in development rates to the blastocyst stage for SCNT embryos from YGR and HGR (33.2% and 35.1%, respec-tively). Pregnancy rates of them were 33.3% and 30.2%, respectively; and birth rates were 16.7% and 11.6%, respectively. (ii) Development rates to the blastocyst stage for SCNT embryos from diffetent individuals (AFB1 and AFB2) differed significantly (27.9% and 39.4%, respectively, P <0.05). Pregnancy rates of them were 36.2% and 36.4%, respectively; and birth rates were 14.9 % and 27.3%, respectively. (iii) There was significant difference in development rates to the blastocyst stage for SCNT embryos from FFB and FOV of the same fetus (37.9% and 41.5%, respectively, P < 0.05). Pregnancy rates of them were 45.7% and 24.1%, respectively; and birth rates were 22.9 % and 10.3%, respectively. Finally, developmental potential of bovine SCNT embryos from all four types of somatic cells from Holstein cows (HGR, AFB, FFB and FOV) were compared. For in vitro development stage, development rates to the blastocyst stage for SCNT embryos from HGR, AFB, FFB and FOV were 35.1%A, 29.4%B, 37.9%A and 41.5%C, respectively (PABC<0.05); for in vivo development stage, pregnancy rates of them were 30.2%, 36.2%, 45.7% and 24.1%, respectively; and birth rates of them were 11.6%, 17.2%, 22.9% and 10.3% respec-tively.
基金the National High Technology Research and Development Program of China (Grant No.2001AA213091) Natural Science Foundation of Hebei Prov-ince (Grant No.C2006001032)
文摘In somatic cell nuclear transfer (SCNT) technologies,the donor cell’s nuclei need to be epigenetically reprogrammed for embryonic development. The incomplete reprogramming of donor cell nuclei has been implicated as a primary reason for the low efficiency of SCNT. DNA methylation is a major epige-netic modification of the genome that regulates crucial aspects of genome function,including estab-lishment of genomic imprinting. In order to make sure whether the DNA methylation reprogramming is efficient in SCNT animals,we analyzed the DNA methylation status of two imprinting genes,H19 and Xist,in lungs of deceased SCNT bovines that died within 48 h of birth using bisulfite sequencing analysis. Our findings demonstrated that cloned bovines showed significantly lower DNA methylation of H19 than controls (P<0.05),and three tested CpGs sites (1,2,3) exhibited unmethylation in one cloned bovine (9C3); however,Xist showed similar DNA methylation levels between clones and con-trols,and both showed hypermethylation (96.11% and 86.67%).
基金This work was supported by the Biology Program of Chinese National Foundation of"863"(Grant No.2001AA213091)by the Major Program of Natural Science Foundation of Beijing(Grant No.5030001).
文摘Cloning by somatic nuclear transfer is an inefficient process in which many of the cloned ani- mals died shortly after birth and displayed organ ab- normalities. In an effort to determine the possible genetic causes of neonatal death and organ abnor- malities, we have examined expression patterns of four genes that modified chromatin (DNMT1, PCAF,MeCP2 and EED) in six organs (heart, liver, spleen, lung, kidney and brain) of both neonatal death cloned bovines (n=9) and normal control calves produced by artificial insemination (AI) using real-time quantitative RT-PCR. The effect of the age of the fibroblast donor cell on the gene expression profiles was also investigated. Aberrant expressions of DNMT1 and PCAF were found in some studied tissues, but the expression of MeCP2 and EED had similar levels to those of the normal controls. The expression of DNMT1 showed a higher level in heart, liver and brain of both cloned bovines. A higher ex- pression level of PCAF was seen in heart and liver of both cloned bovines, but a lower level was seen only in spleen of adult fibroblast (AF) cell-derived clones. Our results suggest that aberrant expression in gene that modified chromatins were found in cloned bovine tissues of neonatal death. Because DNMT1 and PCAF play an important role in DNA methylation and histone acetylation on nuclear chromatin respectively, and normal expression of DNMT1 and PCAF is needed for precious reprogramming of donor nuclear, the aberrant transcription patterns of DNMT1 andPCAF in these clones may contribute to the defects of organs reported in neonatal death of clones.
基金This work was supported by“863” High-Tech Research and Development Program (Grant No. 2001AA213091 & 2002AA206111) Natural Science Foundation of Bejjing (Grant No. 5030001 ).
文摘Somatic cell clone technology is a viable ap- proach to preserving endangered livestock and wildlife ge- netic resources. In the present research, somatic cell nuclear transfer (SCNT) was performed using granulose cells from the critical endangered Chinese red-cross yellow cattle as donor cells. A total of 211 oocytes were manipulated and 166 (79%) of them were successfully enucleated. 112 (67.4%) SCNT embryos were reconstructed, 94 (83%) of them cleaved, and 48 (43%) of them developed to blastocyst stage. SCNT blastocysts were transferred to 6 Holstein recipients, and 2 (33%) of them were found to be pregnant. One of them maintained to term and delivered a calf, whereas another aborted. Effect of different fusion buffer (mannitol vs. Zim- merman fusion buffer) and different activation methods (calcium ionophore+6-DMAP vs. cycloheximide+CB) on fu- sion rate and development of SCNT embryos were investi- gated. The results indicated that: (i) on condition of two DC pulses of 2.5 kV/cm for 10 μs each, fusion rates were higher in mannitol solution than in Zimmerman fusion buffer (71% vs. 61%, respectively, p<0.05), but the blastocysts rates did not differ between two treatments (36% vs. 39%, p>0.05 ); (ii) There was no significant difference in development rates to the blastocyst stage for SCNT embryos activated by calcium ionophore+6-DMAP or by cycloheximide+CB (42% vs. 46%, respectively, p>0.05). Microsatellite DNA analysis examining 28 loci confirmed that the cloned calf was genetically identi- cal to the donor Jinan red-cross yellow cattle and different from the recipient females. Growth and reproductive per- formance of cloned cow were evaluated, and there were no difference i cross-red n it between cloned and normal control Jinan yellow cattle. Furthermore, the cloned yellow cow has delivered a healthy yellow calf.
