Transposon-mediated BAC Transgenesis

动物损失：我们损失了Cre转基因报道鱼品系（tcf 21：CreERT 2，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。 我们需要Cre品系来评估我们的ubb-nucSwitch转基因鱼品系将报告的可信度 不同组织中的Cre活性（目的2.3）。我们也失去了注射了热休克蛋白70的幼年斑马鱼， 含有110 kb BAC转基因的基因座，这些基因座被安置在主要的鱼类设施中。我们需要 用这些鱼来测定tol 2介导的BAC转基因对于BAC跨越 约100 kb的基因组DNA（Aim 2.2）。建立一个从我们的另一个实验室获得的线 自己的设施需要6至8个月（两代鱼）。测试转基因DNA构建体用于 生殖系传播大约需要3 - 5个月。因此，我们估计损失的时间为 在6到8个月之间。 恢复丢失的动物：我们已经询问了产生Cre转基因报告基因的实验室。 再给我们发一次鱼线我们估计，我们有这些鱼在我们的主要设施， 到2013年10月达到繁殖年龄。我们将含有110 kb BAC的hsp 70位点重新注入 预计2013年6月开始筛选转基因创始鱼， 到2013年8月评估转基因率。 试剂损失：由于冷冻，我们失去了sw 105重组工程菌株中的ubb BAC， 冰箱断电需要该菌株来修饰ubb BAC以产生ubbnucSwitch Cre报告基因转基因（Aim 2.2）。BAC转化子的产生和检测 需要一个月左右。 恢复丢失的试剂：我们将ubb BAC重新转换为sw 105 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， 在我们的主要鱼类设施中，成鱼和幼鱼存活率较低。

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.