Transposon-mediated BAC Transgenesis

转座子介导的 BAC 转基因

• 批准号: 8667954
• 负责人: Holger Knaut
• 金额: $ 8.48万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-12-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8667954
• 关键词: Adolescent; Adult; Age; Animal Model; Animals; Bacterial Artificial Chromosomes; Biological Models; Breeding; Cells; Chromosomes, Human, Pair 2; Communities; DNA; Development; Disease; Eye; Fertility; Fishes; Gene Expression Profile; Gene Transfer Techniques; Generations; Genetic; Genetic Engineering; Genome; Genomic DNA; Genomics; Goals; Housing; Injection of therapeutic agent; Length; Libraries; Mediating; Nuclear; Quarantine; Reagent; Recombinants; Recovery; Reporter; Reporting; Research; Research Personnel; Site; Staging; Survival Rate; Techniques; Testing; Time; Tissues; Transgenes; Transgenic Animals; Transgenic Organisms; Transposase; Ubiquitin; Vertebral column; Zebrafish; arm; base; egg; experience; gene function; promoter; research study; restoration; screening; tool; transmission process; zebrafish genome

Animal loss: We lost Cre transgenic reporter fish lines (tcf21:CreERT2, VE-Cdh:Cre, nkx2.5:Cre) that we had obtained from other labs and that were housed in our quarantine room. We need the Cre lines to assess how faithful our ubb-nucSwitch transgenic fish line will report Cre activity in different tissues (Aim 2.3). We also lost juvenile zebrafish injected with the hsp70 locus containing 110 kb BAC transgene that were housed in the main fish facility. We need these fish to determine the efficiency of tol2-mediated BAC transgenesis for BACs spanning about 100 kb of genomic DNA (Aim 2.2). Establishing a line obtained from another lab in our own facility takes 6 to 8 months (two fish generations). Testing transgenic DNA constructs for germline transmission takes about 3 to 5 month. We therefore estimate the time lost to be between 6 to 8 months. Restoration of lost animals: We have asked the labs that generated the Cre transgenic reporter fish lines to send us the lines again. We estimated that we have these fish in our main facility at breeding age by October 2013. We have re-injected the hsp70 locus containing 110 kb BAC transgene and estimate to start screening for transgenic founder fish in June 2013 and finish the assessment of the rate of transgenesis by August 2013. Reagent loss: We lost the ubb BAC in the sw105 recombineering strain due to fridge and freezer power outage. This strain is needed to modify the ubb BAC to generate the ubbnucSwitch Cre reporter transgene (Aim 2.2). The generation and test of BAC transformants takes about a month. Restoration of lost reagents: We have re-transformed the ubb BAC into the sw105 recombineering strain and confirmed the transformants. Time loss: We lost time due to lab closure and recovery efforts, which totals about three months. Additionally, we lack a quarantine room to import fish and are experiencing lower fecundity of adult fish and lower survival rate of baby fish in our main fish facility.

动物损失：我们失去了 Cre 转基因报告鱼系（tcf21:CreERT2、VE-Cdh:Cre、 nkx2.5:Cre)，我们从其他实验室获得并存放在我们的隔离室中。 我们需要 Cre 品系来评估我们的 ubb-nucSwitch 转基因鱼品系报告的准确性 不同组织中的 Cre 活性（目标 2.3）。我们还失去了注射 hsp70 的幼年斑马鱼 含有 110 kb BAC 转基因的基因座，位于主要鱼类设施中。我们需要 这些鱼确定 tol2 介导的 BAC 转基因的效率 约 100 kb 的基因组 DNA（目标 2.2）。建立从我们的另一个实验室获得的生产线 自己的设施需要 6 至 8 个月（两代鱼）。测试转基因 DNA 构建体 种系传播大约需要3至5个月。因此我们估计损失的时间为 6至8个月之间。 恢复丢失的动物：我们询问了产生 Cre 转基因报告基因的实验室 鱼线再次给我们发送鱼线。我们估计我们的主要设施中有这些鱼 2013 年 10 月达到繁殖年龄。我们重新注射了含有 110 kb BAC 的 hsp70 基因座 转基因，预计2013年6月开始筛选转基因创始鱼并完成 2013 年 8 月转基因率评估。 试剂损失：由于冰箱和其他原因，我们在 sw105 重组工程菌株中损失了 ubb BAC 冰箱停电。该菌株需要修改 ubb BAC 以生成 ubbnucSwitch Cre 报告基因转基因（目标 2.2）。 BAC转化子的产生和测试 大约需要一个月的时间。 丢失试剂的恢复：我们将ubb BAC重新改造为sw105 重组工程菌株并确认转化子。 时间损失：由于实验室关闭和恢复工作，我们损失了大约三个月的时间。 此外，我们缺乏进口鱼类的检疫室，并且鱼类的繁殖力较低 在我们的主要养鱼设施中，成鱼和幼鱼的成活率较低。