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.
Transgenic zebrafish models are essential for our understanding of gene function in normal development and disease. While transgenesis of small DNA constructs is a standard technique in zebrafish; the integration of 100 kilo bases or more into the zebrafish genome is hampered by its low efficiency of transgenesis. In this proposal; we intend to develop a technique for efficient integration of large transgenes into the zebrafish genome.
|Fuentes, Fernando; Reynolds, Eric; Lewellis, Stephen W et al. (2016) A Plasmid Set for Efficient Bacterial Artificial Chromosome (BAC) Transgenesis in Zebrafish. G3 (Bethesda) 6:829-34|
|Nagelberg, Danielle; Wang, Jinhu; Su, Rina et al. (2015) Origin, Specification, and Plasticity of the Great Vessels of the Heart. Curr Biol 25:2099-110|