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转基因记者鱼线（TCF21：Creert2，VE-CDH：CRE，CRE， 我们从其他实验室获得的NKX2.5：CRE），这些实验室位于我们的隔离室。 我们需要CRE线来评估我们的UBB-Nucswitch转基因鱼类系列的忠诚 在不同组织中的CRE活性（AIM 2.3）。我们还失去了少年斑马鱼注入了HSP70 含有110 kb BAC转基因的基因座，该基因烯位于主要鱼类设施中。我们需要 这些鱼以确定跨越BAC的TOL2介导的BAC转基因的效率 约100 kb的基因组DNA（AIM 2.2）。建立从我们的另一个实验室获得的线路 自己的设施需要6到8个月（两个鱼世代）。测试转基因DNA构建体 种系传输大约需要3到5个月。因此，我们估计失去的时间 在6到8个月之间。 恢复失落的动物：我们询问了产生CRE转基因记者的实验室 鱼线再次向我们发送线。我们估计我们在我们的主要设施中有这些鱼 到2013年10月，繁殖年龄。我们已重新注射包含110 kb BAC的HSP70基因座 Transgene和估计将于2013年6月开始对转基因创始人鱼进行筛查，并完成 到2013年8月的转基因率评估。 试剂损失：由于冰箱和 冰柜电力中断。需要这种菌株来修改UBB BAC以产生Ubbnucswitch CRE报告基因转基因（AIM 2.2）。 BAC转化体的产生和测试 大约需要一个月。 恢复丢失的试剂：我们已将UBB BAC重新转换为SW105 重组应变并确认转化体。 时间损失：由于实验室关闭和恢复工作，我们浪费了时间，总计约三个月。 此外，我们缺少隔离房间进口鱼，并且经历了较低的繁殖力 我们主要鱼类设施中的成年鱼和较低的婴儿鱼的存活率。