Regulated Gene Expression in the Chicken Embryo by Using Replication-Competent Retroviral Vectors

Abstract

Rous sarcoma virus (RSV)-derived retroviral vector could efficiently deliver the green fluorescent protein (GFP), which is driven by the internal cytomegalovirus enhancer/promoter, into restricted cell populations in the chicken embryo. RSV-derived vectors coupled with the tet regulatory elements also revealed doxycyclinedependent inducible GFP expression in the chicken embryo in ovo.

Distinct susceptibility of developing neurons to death following Bax overexpression in the chicken embryo

“…Accordingly, to introduce Bax specifically into MNs during the period of early PCD, we have used in ovo electroporation 26 coupled with the tet regulatory system. 23 To target gene expression to spinal MNs, the reverse tetracycline-controlled transactivator is expressed under the control of the MN-specific HB9 promoter. 27 We successfully controlled GFP expression spatially and temporally in MNs by administration of doxycycline (see Figure S4 in the supplementary material).…”

“…High-titer viruses were generated by standard procedures with only minor modification. 15,23 Briefly, virus propagation was initiated by transfection of retroviral plasmid constructs into the DF-1 chick fibroblast cell line. 16,17 After several passages of DF-1 cells, the supernatants were collected and concentrated by centrifugation at 22 000 rpm for 3 h in a Beckman SW 28 rotor and stored in aliquots at À801C.…”

“…DF-1 cells were infected with each of the viruses at 50 infectious units per cell. After a few passages of the DF-1 cells, immunoblotting was performed as described previously, 23 using the polyclonal anti-Bax antibody N-20 (Santa Cruz). For cell growth assays, cells were initially plated at a density of 6 Â 10 3 cells/well in 12-well tissue culture plates (Greiner).…”

“…38 Viruses were injected into embryos at stage 9 (E1.5) via a pulled micropipette needle as described. 15,23 Typically, a total of 0.2-0.5 ml of virus solution was injected into a lumen of the neural tube.…”

“…15,23,34 Primary antibodies used in this study were the anti-Islet-1/2 (clone 4D5, Developmental Studies Hybridoma Bank, DSHB), anti-Lim-3 (clone 67.4E12, DSHB), antiviral p19 GAG protein (clone AMV-3C2, DSHB), antiactivated caspase-3 (clone C92-605, Pharmingen), and anti-Bax (clone 4F11, MBL). We tested three distinct antibodies for hBax from either Santa Cruz (polyclonal antibody, N-20), MBL (monoclonal antibody, 4F11), or Pharmingen (polyclonal antibody).…”