目的探讨应用单克隆接种/成像设备VIPS(Verified In-situ Plate Seeding)建立CHO工程细胞株单克隆筛选的方法。方法采用VIPS将稳定转染目的基因的细胞池Cell pool 1分别于含不同组合(共18种组合,编号1~18)及体积(100和200μL/孔)培养基...目的探讨应用单克隆接种/成像设备VIPS(Verified In-situ Plate Seeding)建立CHO工程细胞株单克隆筛选的方法。方法采用VIPS将稳定转染目的基因的细胞池Cell pool 1分别于含不同组合(共18种组合,编号1~18)及体积(100和200μL/孔)培养基的96孔板中进行单细胞接种,并进行单克隆源性追踪拍照,根据图像结果,计算单克隆接种率、克隆形成率、单克隆增殖速率,评价单克隆源性图像效果。通过稳定转染目的基因的细胞池Cell pool 2、Cell pool 3、Cell pool 4验证该方法的适用性。结果最适单克隆培养基为MediumⅡ,培养基体积为100μL/孔。最佳工艺条件下,Cell pool 1的单克隆接种率为80%,克隆形成率为83%,增殖速率较快,单克隆源性图像清晰。Cell pool 2、Cell pool 3、Cell pool 4在最佳工艺条件下,单克隆接种率均值为78%,克隆形成率均值为67%,增殖速率略有差异,细胞分裂过程图像均清晰。结论采用VIPS建立的CHO工程细胞株单克隆筛选方法可提高CHO工程细胞株的克隆形成率,且能够提供充分的单克隆源性证明。展开更多
Background Bt technology has played significant role in controlling bollworms and increasing cotton yield in earlier days of its introduction,a subsequent decline in yield became apparent over time.This decline may be...Background Bt technology has played significant role in controlling bollworms and increasing cotton yield in earlier days of its introduction,a subsequent decline in yield became apparent over time.This decline may be attributed to various environmental factors,pest dynamics,or combination of both.Therefore,the present biophysical survey and questionnaire were designed to evaluate the impact of Bt cotton on bollworms management and its effect on reducing spray costs,targeting farmers with varied landholdings and educational backgrounds.Additionally,data on farmers'cultivated varieties and the prevalence of bollworms and sucking insects in their fields were recorded.Subsequently,about eleven thousand cotton samples from farmer fields were tested for Cry1Ac,Cry2Ab and Vip3A genes by strip test.Results In this analysis,83% of the farmers planting approved varieties believe that Bt technology control bollworms,while 17% hold contradictory views.Similarly,among farmers cultivating unapproved varieties,77% agree on effectiveness of Bt technology against bollworms,while 23% disagree.On the other hand,67% of farmers planting approved varieties believe that Bt technology does not reduce spray costs,while 33% agree with the effectiveness.Similarly,78% of farmers cultivating unapproved varieties express doubt regarding its role to reduce spray costs,while 22% are in favour of this notion.Differences in opinions on the effectiveness of Bt cotton in controlling bollworms and reducing spray cost between farmers planting unapproved and approved varieties may stem from several factors.One major cause is the heavy infestation of sucking insects,which is probably due to the narrow genetic variation of the cultivated varieties.Additionally,the widespread cultivation of unapproved varieties(21.67%)is also an important factor to cause different opinions on the effectiveness of Bt cotton.Conclusion Based on our findings,we propose that the ineffective control of pests on cotton crop may be attributed to large scale cultivation of unapproved varieties and non-inclusion of double and triple transgene technologies in country's sowing plan.On the basis of our findings,we suggest cotton breeders,regulatory bodies and legislative bodies to discourage the cultivation of unapproved varieties and impure seed.Moreover,the adoption of double and triple Bt genes in cottons with a broad genetic variation could facilitate the revival of the cotton industry,and presenting a promising way forward.展开更多
文摘目的探讨应用单克隆接种/成像设备VIPS(Verified In-situ Plate Seeding)建立CHO工程细胞株单克隆筛选的方法。方法采用VIPS将稳定转染目的基因的细胞池Cell pool 1分别于含不同组合(共18种组合,编号1~18)及体积(100和200μL/孔)培养基的96孔板中进行单细胞接种,并进行单克隆源性追踪拍照,根据图像结果,计算单克隆接种率、克隆形成率、单克隆增殖速率,评价单克隆源性图像效果。通过稳定转染目的基因的细胞池Cell pool 2、Cell pool 3、Cell pool 4验证该方法的适用性。结果最适单克隆培养基为MediumⅡ,培养基体积为100μL/孔。最佳工艺条件下,Cell pool 1的单克隆接种率为80%,克隆形成率为83%,增殖速率较快,单克隆源性图像清晰。Cell pool 2、Cell pool 3、Cell pool 4在最佳工艺条件下,单克隆接种率均值为78%,克隆形成率均值为67%,增殖速率略有差异,细胞分裂过程图像均清晰。结论采用VIPS建立的CHO工程细胞株单克隆筛选方法可提高CHO工程细胞株的克隆形成率,且能够提供充分的单克隆源性证明。
基金supported by Punjab Agriculture Research Board,Grant numbers PARB No.890received research support from Punjab Agriculture Board。
文摘Background Bt technology has played significant role in controlling bollworms and increasing cotton yield in earlier days of its introduction,a subsequent decline in yield became apparent over time.This decline may be attributed to various environmental factors,pest dynamics,or combination of both.Therefore,the present biophysical survey and questionnaire were designed to evaluate the impact of Bt cotton on bollworms management and its effect on reducing spray costs,targeting farmers with varied landholdings and educational backgrounds.Additionally,data on farmers'cultivated varieties and the prevalence of bollworms and sucking insects in their fields were recorded.Subsequently,about eleven thousand cotton samples from farmer fields were tested for Cry1Ac,Cry2Ab and Vip3A genes by strip test.Results In this analysis,83% of the farmers planting approved varieties believe that Bt technology control bollworms,while 17% hold contradictory views.Similarly,among farmers cultivating unapproved varieties,77% agree on effectiveness of Bt technology against bollworms,while 23% disagree.On the other hand,67% of farmers planting approved varieties believe that Bt technology does not reduce spray costs,while 33% agree with the effectiveness.Similarly,78% of farmers cultivating unapproved varieties express doubt regarding its role to reduce spray costs,while 22% are in favour of this notion.Differences in opinions on the effectiveness of Bt cotton in controlling bollworms and reducing spray cost between farmers planting unapproved and approved varieties may stem from several factors.One major cause is the heavy infestation of sucking insects,which is probably due to the narrow genetic variation of the cultivated varieties.Additionally,the widespread cultivation of unapproved varieties(21.67%)is also an important factor to cause different opinions on the effectiveness of Bt cotton.Conclusion Based on our findings,we propose that the ineffective control of pests on cotton crop may be attributed to large scale cultivation of unapproved varieties and non-inclusion of double and triple transgene technologies in country's sowing plan.On the basis of our findings,we suggest cotton breeders,regulatory bodies and legislative bodies to discourage the cultivation of unapproved varieties and impure seed.Moreover,the adoption of double and triple Bt genes in cottons with a broad genetic variation could facilitate the revival of the cotton industry,and presenting a promising way forward.
