The Earth does not revolve around the Sun. A two-part list of links to download the article, or parts of the article, in various formats. At least, this is the model you are using." Indeed, we observed that the membrane-bound GFP results in a better resolution of cell membrane processes (protrusions, filopodia, etc.) Transgenic embryos were dissected and fixed for 1 hr in 4% formaldehyde. Compared to the existing quail lines carrying ubiquitously expressed fluorescent proteins, this line should prove useful to researchers in the field. (A–D) Cross-section of the head of an E4 embryo, electroporated one day earlier in the optic cup with a CrystallGFP minigene. The method consists of just injecting a mixture of the plasmid together with lipofectamine into the circulating blood stream of quail embryos at about E2.5, where the construct transfects some of the circulating primordial germ cells (PGCs). Please make a tax-deductible donation if you value independent science communication, collaboration, participation, and open access. The Australian Regenerative Medicine Institute is supported by grants from the State Government of Victoria and the Australian Government. This makes it due to its short 6 week lifecycle and small size highly suitable for avian transgenesis experiments. Indeed, all too often it seems that the research potential of a new molecular technique is overshadowed by its oh-so-exciting clinical applications. Given the current world situation, we will give authors as much time as they need to submit revised manuscripts. Expression of GFP or mCherry was visible in the beak, eyes and legs of the transgenic birds compared to wild-type animals (Figure 1G,H). Others decided to set up collaborations with the laboratories that generated and maintain the lines, which regularly provide fertilized eggs for their experiments. Although it is technically possible to genome-edit the chicken, its long generation time (6 months to sexual maturity) makes it an impractical lab model and has prevented it widespread use in research. To test whether the direct transfection of PGCs can be achieved in quails, we determined whether we could observe PGCs transfected with fluorescent proteins once they colonized the gonads. 1 μl of injection mix was injected in the dorsal aorta of 2.5-day-old embryos. We designed a muscle-specific promoter, based on a synthetic reporter derived from the MLC1F/3F gene regulatory sequences previously utilized for mouse transgenesis (3F-nlacZ-E; Kelly et al., 1995). This construct was shown to drive strong LacZ expression in all (head and body) striated muscles from the early steps of myogenesis in somites of mouse embryos throughout embryogenesis, as well as in all skeletal muscles of the foetus and in the adult (Kelly et al., 1995). Plasmid DNA was purified using Nucleobond Xtra Midi EF kit. The zygote is very difficult to access as it initiates its development internally, in the hen's oviduct and on a large yolk. We confirmed that these were PGCs using whole-mount immunostaining with a PGC-specific marker VASA (Figure 1C–F). The direct injection technique is performed as described in Tyack et al., 2013. (B) Gonads from E7 embryo dissected 5 days after in vivo PGC transfection, showing GFP-positive transfected PGCs (arrowheads). Because of this, most researchers in avian genetics have focused their efforts on two distinct methods (Nishijima and Iijima, 2013): i) the genetic manipulation of primordial germ cells (PGCs) in vitro, which are injected back into recipient embryos (Idoko-Akoh et al., 2018; Park et al., 2014; Taylor et al., 2017; van de Lavoir et al., 2006) or ii) the direct infection of PGCs within the subgerminal cavity with replication-defective lentiviruses (Bosselman et al., 1989; McGrew et al., 2004). Moreover, we present a community website where existing lines are described and new ones can be uploaded. Although hypoxic or ischemia tolerance in AGS involves physiological adaptations, little is known about the critical cellular mechanisms underlying intrinsic AGS cell resilience to metabolic stress. Upon request, the scripts/database structure can be released through a git repository so that anyone can contribute. Avian transgenesis is so much more difficult than in mammals or Xenopus. the type of line and the method used to generate it, its availability, etc.). Whatever. As an aid to transgenic quail research, QuailNet integrates various resources: The 3D quail anatomy portal, which contains 3D models of quail embryos covering a range of development stages from embryonic day E1 (HH7) to E11 (HH40). The transgenesis efficiency (~1%) is manageable, especially when combined with rapid screening techniques, including such as the here developed eye lens specific crystallGFP. We have not observed any significant decrease of the signal over time, suggesting, as mentioned in the text that Tol2 insertions are not particularly prone to inactivation over time. How is this going to be curated? Chicken PGCs are transfected during their transient journey in the bloodstream, using a transfection mix containing lipofectamine, a transgenesis plasmid with Tol2 elements flanking the DNA construct to be inserted and a plasmid coding for transposase under … GFP) are clearly out of size range for viruses. Similar to chicken, quail lay about one egg per day, and their smaller size makes them more compatible with the frequently limited space of animal facilities. 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