Genetically modified wheat has not been commercially utilized in agriculture largely due to regulatory hurdles associated with traditional transformation methods. Development of marker-free transgenic wheat plants wil...Genetically modified wheat has not been commercially utilized in agriculture largely due to regulatory hurdles associated with traditional transformation methods. Development of marker-free transgenic wheat plants will help to facilitate biosafety evaluation and the eventual environmental release of transgenic wheat varieties. In this study, the marker-free transgenic wheat plants previously obtained by Agrobacterium-mediated co-transformation of double T-DNAs vector were identified by fluorescence in situ hybridization(FISH) in the T1 generation, and their genetic stability and agronomic traits were analyzed in T2 and T3 generations. FISH analysis indicated that the transgene often integrated into a position at the distal region of wheat chromosomes. Furthermore, we show that the GUS transgene was stably inherited in the marker-free transgenic plants in T1 to T3 generations. No significant differences in agronomic traits or grain characteristics were observed in T3 generation, with the exception of a small variation in spike length and grains per spike in a few lines. The selection marker of bar gene was not found in the transgenic plants through T1 to T3 generations. The results from this investigation lay a solid foundation for the potential application of the marker-free transgenic wheat plants achieved through the co-transformation of double T-DNAs vector by Agrobacterium in agriculture after biosafty evaluation.展开更多
Objective: Embryonic chromosomal abnormality is one of the main reasons for in vitro fertilization (IVF) failure. This study aimed at evaluating the value of Fluorescence in-situ Hybridization (FISH)-based Preimplanta...Objective: Embryonic chromosomal abnormality is one of the main reasons for in vitro fertilization (IVF) failure. This study aimed at evaluating the value of Fluorescence in-situ Hybridization (FISH)-based Preimplantation Genetic Diagnosis (PGD) in screening for embryonic chromosomal abnormality to increase the successful rate of IVF. Method: Ten couples, four with high risk of chromosomal abnormality and six infertile couples, underwent FISH-based PGD during IVF procedure. At day 3, one or two blastomeres were aspirated from each embryo. Biopsied blastomeres were examined using FISH analysis to screen out embryos with chromosomal abnormalities. At day 4, embryos without detectable chromosomal abnormality were transferred to the mother bodies as in regular IVF. Results: Among 54 embryos screened using FISH-based PGD, 30 embryos were detected to have chromosomal abnormalities. The 24 healthy embryos were implanted, resulting in four clinical pregnancies, two of which led to successful normal birth of two healthy babies; one to ongoing pregnancy during the writing of this article; and one to ectopic pregnancy. Conclusion: FISH-based PGD is an effective method for detecting embryonic chromosomal abnormality, which is one of the common causes of spontaneous miscarriages and chromosomally unbalanced offsprings.展开更多
目的运用荧光原位杂交技术(fluorescent in situ hybridization,FISH)对废弃胚胎固定好的单卵裂球进行检测,了解染色体数目异常,即非整倍体(anenploidy)改变在体外受精(in vitro-fertilization,IVF)中的发生情况。方法应用橘红色LSI-21...目的运用荧光原位杂交技术(fluorescent in situ hybridization,FISH)对废弃胚胎固定好的单卵裂球进行检测,了解染色体数目异常,即非整倍体(anenploidy)改变在体外受精(in vitro-fertilization,IVF)中的发生情况。方法应用橘红色LSI-21探针对IVF患者正常受精D3的废弃胚胎活检后单个卵裂球进行FISH检测,统计并分析其信号结果。结果活检胚胎33个,2个胚胎未出现信号,获31个胚胎,共64个卵裂球杂交信号。包括年龄≥35岁患者胚胎7个,<35岁患者胚胎24个。其中2倍体信号胚胎20个,单倍体信号胚胎3个,3倍体信号胚胎8个,21号染色体异常的胚胎为11个,其中35岁以上患者占3个,35岁以下患者占8个。结论人IVF废弃胚胎21号染色体异常几率多。患者年龄≥35岁者尤甚。展开更多
In order to establish a simple and useful way for preimplantation genetic diagnosis (PGD) of chromosomal diseases in general IVF laboratory, the methods that are most commonly used in the embryo biopsy, fixation of bl...In order to establish a simple and useful way for preimplantation genetic diagnosis (PGD) of chromosomal diseases in general IVF laboratory, the methods that are most commonly used in the embryo biopsy, fixation of blastomere and fluorescence in situ hybridization were compared. The three aspects of PGD were analyzed respectively. There was no significant difference in further de- velopment capacity of embryos between mechanical (79.7%) and chemical biopsy group (78.6%) (P>0.05). In this study, more cells were successfully fixed with the Tween/HCL method (93.8%) than with the methanol/acetic acid method (80.5%, P<0.05). There was no significant difference in cyto- plasm remains between methanol/acetic acid method and Tween/HCL method (P>0.05). The hy- bridization efficiency of fluorescence in situ hybridization was 89.5% in successive denaturation method and 90.9% in codenaturation method with the difference being not significant (P>0.05). In conclusion, the mechanical or chemical method, Tween/HCL fixation method and codenaturation fluorescence in situ hybridization method can constitute a simple and useful way for PGD of chro- mosomal diseases.展开更多
基金the Ministry of Agriculture of China for the National Transgenic Research Program (2016ZX08010004)the Chinese Academy of Agricultural Sciences for the Agricultural Science and Technology Innovation Program (ASTIP-2060302-2-19)
文摘Genetically modified wheat has not been commercially utilized in agriculture largely due to regulatory hurdles associated with traditional transformation methods. Development of marker-free transgenic wheat plants will help to facilitate biosafety evaluation and the eventual environmental release of transgenic wheat varieties. In this study, the marker-free transgenic wheat plants previously obtained by Agrobacterium-mediated co-transformation of double T-DNAs vector were identified by fluorescence in situ hybridization(FISH) in the T1 generation, and their genetic stability and agronomic traits were analyzed in T2 and T3 generations. FISH analysis indicated that the transgene often integrated into a position at the distal region of wheat chromosomes. Furthermore, we show that the GUS transgene was stably inherited in the marker-free transgenic plants in T1 to T3 generations. No significant differences in agronomic traits or grain characteristics were observed in T3 generation, with the exception of a small variation in spike length and grains per spike in a few lines. The selection marker of bar gene was not found in the transgenic plants through T1 to T3 generations. The results from this investigation lay a solid foundation for the potential application of the marker-free transgenic wheat plants achieved through the co-transformation of double T-DNAs vector by Agrobacterium in agriculture after biosafty evaluation.
文摘Objective: Embryonic chromosomal abnormality is one of the main reasons for in vitro fertilization (IVF) failure. This study aimed at evaluating the value of Fluorescence in-situ Hybridization (FISH)-based Preimplantation Genetic Diagnosis (PGD) in screening for embryonic chromosomal abnormality to increase the successful rate of IVF. Method: Ten couples, four with high risk of chromosomal abnormality and six infertile couples, underwent FISH-based PGD during IVF procedure. At day 3, one or two blastomeres were aspirated from each embryo. Biopsied blastomeres were examined using FISH analysis to screen out embryos with chromosomal abnormalities. At day 4, embryos without detectable chromosomal abnormality were transferred to the mother bodies as in regular IVF. Results: Among 54 embryos screened using FISH-based PGD, 30 embryos were detected to have chromosomal abnormalities. The 24 healthy embryos were implanted, resulting in four clinical pregnancies, two of which led to successful normal birth of two healthy babies; one to ongoing pregnancy during the writing of this article; and one to ectopic pregnancy. Conclusion: FISH-based PGD is an effective method for detecting embryonic chromosomal abnormality, which is one of the common causes of spontaneous miscarriages and chromosomally unbalanced offsprings.
文摘目的运用荧光原位杂交技术(fluorescent in situ hybridization,FISH)对废弃胚胎固定好的单卵裂球进行检测,了解染色体数目异常,即非整倍体(anenploidy)改变在体外受精(in vitro-fertilization,IVF)中的发生情况。方法应用橘红色LSI-21探针对IVF患者正常受精D3的废弃胚胎活检后单个卵裂球进行FISH检测,统计并分析其信号结果。结果活检胚胎33个,2个胚胎未出现信号,获31个胚胎,共64个卵裂球杂交信号。包括年龄≥35岁患者胚胎7个,<35岁患者胚胎24个。其中2倍体信号胚胎20个,单倍体信号胚胎3个,3倍体信号胚胎8个,21号染色体异常的胚胎为11个,其中35岁以上患者占3个,35岁以下患者占8个。结论人IVF废弃胚胎21号染色体异常几率多。患者年龄≥35岁者尤甚。
文摘In order to establish a simple and useful way for preimplantation genetic diagnosis (PGD) of chromosomal diseases in general IVF laboratory, the methods that are most commonly used in the embryo biopsy, fixation of blastomere and fluorescence in situ hybridization were compared. The three aspects of PGD were analyzed respectively. There was no significant difference in further de- velopment capacity of embryos between mechanical (79.7%) and chemical biopsy group (78.6%) (P>0.05). In this study, more cells were successfully fixed with the Tween/HCL method (93.8%) than with the methanol/acetic acid method (80.5%, P<0.05). There was no significant difference in cyto- plasm remains between methanol/acetic acid method and Tween/HCL method (P>0.05). The hy- bridization efficiency of fluorescence in situ hybridization was 89.5% in successive denaturation method and 90.9% in codenaturation method with the difference being not significant (P>0.05). In conclusion, the mechanical or chemical method, Tween/HCL fixation method and codenaturation fluorescence in situ hybridization method can constitute a simple and useful way for PGD of chro- mosomal diseases.
