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Multiplexed fluorescent hybridization chain reaction (HCR) and advanced imaging techniques can be used to evaluate combinatorial gene expression patterns in whole mouse embryos with unprecedented spatial resolution. Using HCR, DNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled DNA HCR hairpins self-assemble into tethered fluorescent amplification polymers. Each target mRNA is detected by a probe set containing one or more DNA probes, with each probe carrying two HCR initiators. For multiplexed experiments, probe sets for different target mRNAs carry orthogonal initiators that trigger orthogonal DNA HCR amplification cascades labeled by spectrally distinct fluorophores. As a result, in situ amplification is performed for all targets simultaneously, and the duration of the experiment is independent of the number of target mRNAs. We have used multiplexed fluorescent in situ HCR and advanced imaging technologies to address questions of cell heterogeneity and tissue complexity in craniofacial patterning and anterior neural development. In the sample protocol presented here, we detect three different mRNA targets: Tg(egfp), encoding the enhanced green fluorescent protein (GFP) transgene (typically used as a control); Twist1, encoding a transcription factor involved in cell lineage determination and differentiation; and Pax2, encoding a transcription factor expressed in the mid-hindbrain region of the mouse embryo., (© 2015 Cold Spring Harbor Laboratory Press.)

This weeklong protocol for making and testing lentivirus has been used in the Advanced Topics in Molecular Neuroscience (ATMN) lecture and laboratory course at Cold Spring Harbor Laboratory (CSHL) for nearly a decade. Lentiviruses are derived from HIV-1 and are ideal vehicles for the delivery of multiple genes of interest into target cells. In this protocol, 2A peptide-linked sequences are used to create a bicistronic lentiviral construct containing a ubiquitous promoter (chick β actin with a cytomegalovirus [CMV] early enhancer) driving dual expression of two fluorescent proteins (FP): H2B-Cerulean (a nuclear-localized blue FP) and Dendra2 (a photoactivatable green FP that converts to red after exposure to UV light). Polymerase chain reaction (PCR) amplification of the bicistronic insert is followed by subcloning into a lentiviral vector and transfection into a packaging cell line. The resulting viral supernatants can be used to prepare concentrated stocks and infect cells for imaging via epifluorescent and confocal microscopy., (© 2014 Cold Spring Harbor Laboratory Press.)

The ability to generate transgenic mice has been a powerful tool in studying functional genomics, and much of our knowledge about developmental biology has come from the study of chicken embryology. Unfortunately, the availability of molecular genetic techniques, such as transgenics and knockouts, has been limited for developmental biologists using avian animal models. Efforts to develop a system for the rapid production of transgenic chickens have met with many obstacles, including high animal husbandry costs and long generational times. Recently, the Japanese quail has proven to be an excellent model organism for the production of transgenic avians using lentiviral vectors. The relatively small size of the adults, short time to sexual maturity, and prodigious egg production of the Japanese quail make development of transgenic lines less labor- and space-intensive compared to chickens. The high degree of homology between chicken and quail genomes allows researchers to design highly specific DNA constructs for the production of transgenic birds. In addition, transgenic quail offer all of the advantages of the classic avian developmental model system, such as the ability to readily produce quail:chick transplant chimeras. Finally, Japanese quail are ideal for in ovo imaging of embryos expressing fluorescent reporters introduced from a transgene and/or electroporation. Here, we provide detailed methods for generating transgenic quail using high-titer lentivirus.

After mosaic founder breeding pairs of Japanese quail start to produce fertile eggs, the hatchlings must be screened for germ-line transmission to the subsequent G1 generation. This article describes how to isolate hatchling genomic DNA from the chorioallantoic membrane (CAM), which remains inside the egg after hatching. Collecting genomic DNA from the CAM decreases the hatchling's stress during handling and eliminates the need for a blood draw. By following this protocol, the CAM of a single egg will provide 50 microg or more of high-quality genomic DNA. The article also describes how to screen the genomic DNA samples for the transgene by the polymerase chain reaction (PCR). PCR genotyping should be used for screening hatchlings with a nonfluorescent transgene or with a fluorescently labeled transgene that does not lend itself well to phenotypic screening.

This protocol describes how to generate transgenic quail by injecting a lentiviral vector into freshly laid Japanese quail eggs at stage X. The lentivirus infects primordial germ cells originating in the area pellucida.

This protocol describes how to generate high-titer lentivirus for the production of transgenic Japanese quail. The virus is pseudotyped with vesicular stomatitis virus with the envelope G glycoprotein (VSV-g), which gives a broad infectious range and allows concentration of viral supernatants by ultracentrifugation. Using this method, we typically produce titers >1 x 10(8) transforming units (TU)/mL and recommend using a virus with a titer at least this high for in vivo work.