Journal: BMC Developmental Biology

Article Title: Endogenous expression of ASLV viral proteins in specific pathogen free chicken embryos: relevance for the developmental biology research field

doi: 10.1186/1471-213X-10-106

Figure Lengend Snippet: ASLV genomic organization and target sites for primers used in this study . This diagram represents the generic organization of ASLV viruses for the gag (blue) pol (green) and env (grey) genes and their respective subunits for proteins p19, p2, p10, p27, NCp12, PRp15, reverse transcriptase, integrase and gp85. Target sites for the primers are indicated by green triangles, dark green for forward and light green for reverse primers. The U1, U2 and P27 primer sets detect all viral subgroups, while the E primer set binds only to viruses of the E subgroup. Since differences exist between viral subgroups and even within members of the same subgroup this diagram is not drawn to scale.

Article Snippet: Antibodies and dilutions used were as follows: AMV-3C2 mouse monoclonal antibody raised against the ASLV viral matrix protein p19 (1:5; NICHD-funded Developmental Studies Hybridoma Bank maintained by The University of Iowa, Iowa City, IA); p27 rabbit polyclonal antibody raised against the ASLV viral capsid protein p27 (1:1000; Charles River Laboratories, Wilmington, MA).

Techniques: Reverse Transcription

Journal: BMC Developmental Biology

Article Title: Endogenous expression of ASLV viral proteins in specific pathogen free chicken embryos: relevance for the developmental biology research field

doi: 10.1186/1471-213X-10-106

Figure Lengend Snippet: Immunohistochemical detection of ALV viral proteins in certified SPF chicken embryos . (A-D) Transversal sections of ED6 chick embryo heads, processed by IHC against p27 viral protein (A and C) and DAPI (B and D). (A-B) Section corresponding to an embryo from Flock 1 that was negative for endogenous viral protein expression. (C-D) Section corresponding to a similar region of another embryo from Flock 1, showing extensive expression of p27 viral protein in the retinal neuroepithelium (rn), surrounding cephalic mesenchyme (cm) and brain neuroepithelium (bn) (vc: vitreal cavity). (E and F) Transversal sections of the retina of two different ED11 embryos from Flock 4 showing discrete columns of p27 positive cells; notice in F that in some cases only a few cells are positive for endogenous viral proteins (onl: outer nuclear layer; inl: inner nuclear layer; gcl: ganglion cell layer). (G) Transversal section through the lens of an ED6 embryo from Flock 2 showing endogenous expression of p27 viral protein (le: lens; dc: developing cornea). (H) Transversal section of the retina of an ED6 embryo from Flock 1 showing co-expression of p19 (red) and p27 (green) (rn: retinal neuroepithelium; vc: vitreal cavity). Notice the columnar pattern of expression, particularly in the retinal neuroepithelium and the lens. (I-J) Transversal section through the brain of an ED11 embryo from Flock 4; (I) bright field image; (J) immunostaining for p27 viral protein. Small, well defined areas of the developing brain (br) showed positive expression of p27 (arrows); these areas showed a symmetrical pattern, being present in both, right and left brain structures (vc: ventricular cavity). Scale bar in (J) corresponds to 150 μm for A-D; 100 μm for E-F and H; 250 μm for G and 450 μm for I-J.

Article Snippet: Antibodies and dilutions used were as follows: AMV-3C2 mouse monoclonal antibody raised against the ASLV viral matrix protein p19 (1:5; NICHD-funded Developmental Studies Hybridoma Bank maintained by The University of Iowa, Iowa City, IA); p27 rabbit polyclonal antibody raised against the ASLV viral capsid protein p27 (1:1000; Charles River Laboratories, Wilmington, MA).

Techniques: Immunohistochemical staining, Expressing, Immunostaining

Journal: BMC Developmental Biology

Article Title: Endogenous expression of ASLV viral proteins in specific pathogen free chicken embryos: relevance for the developmental biology research field

doi: 10.1186/1471-213X-10-106

Figure Lengend Snippet: Detection of infective ASLV viral particles in amniotic fluid from SPF chicken embryos . ( A-D) Inoculation assay for detection of exogenous ASLV infection . DF-1 chicken fibroblast cells cultured in the presence of (A) no amniotic fluid; (B) amniotic fluid from untreated SPF chicken embryo; and (C) amniotic fluid from SPF chicken embryo infected with RCAS-nucGFP construct. Inoculated cells were processed for immunohistochemical detection of ASLV viral protein p27 (red) and counterstained with DAPI (blue). Lack of immunohistochemical signal in cells inoculated with amniotic fluid from SPF untreated embryos (B) suggested that SPF embryos were not pre-infected with exogenous ASLV virus. (A) and (C) were used as negative and positive controls respectively. af: amniotic fluid. Scale bar:20 μm. (D). RT-PCR analysis of supernatants from (-) non-inoculated DF-1 cells; or DF-1 cells inoculated with (+) amniotic fluid from SPF embryos infected with RCAS-nucGPF; or (1-3) untreated SPF embryos, confirmed absence of exogenous viral infection in SPF embryos. (E) Detection of endogenous infective viral particles directly from amniotic fluid . Amniotic fluid from (+) SPF embryos infected with RCAS-nucGPF; or (1) untreated SPF embryos negative for p19/p27 by IHC; or (2-3) untreated SPF embryos positive for p19/p27 by IHC, was used for direct RT-PCR detection of infective ASLV viral particles. Positive RT-PCR amplification from amniotic fluid from embryos purposely infected with RCAS-nucGFP (+) set proof-of-principle for the approach. On the other hand, lack of RT-PCR amplification in amniotic fluid from SPF untreated embryos further demonstrated absence of both, exogenous and endogenous ASLV infective viral particles. (-) RT-PCR negative control.

Article Snippet: Antibodies and dilutions used were as follows: AMV-3C2 mouse monoclonal antibody raised against the ASLV viral matrix protein p19 (1:5; NICHD-funded Developmental Studies Hybridoma Bank maintained by The University of Iowa, Iowa City, IA); p27 rabbit polyclonal antibody raised against the ASLV viral capsid protein p27 (1:1000; Charles River Laboratories, Wilmington, MA).

Techniques: Infection, Cell Culture, Construct, Immunohistochemical staining, Virus, Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control

Journal: BMC Developmental Biology

Article Title: Endogenous expression of ASLV viral proteins in specific pathogen free chicken embryos: relevance for the developmental biology research field

doi: 10.1186/1471-213X-10-106

Figure Lengend Snippet: Detection of genomic ev loci regions in SPF embryos by PCR

Article Snippet: Antibodies and dilutions used were as follows: AMV-3C2 mouse monoclonal antibody raised against the ASLV viral matrix protein p19 (1:5; NICHD-funded Developmental Studies Hybridoma Bank maintained by The University of Iowa, Iowa City, IA); p27 rabbit polyclonal antibody raised against the ASLV viral capsid protein p27 (1:1000; Charles River Laboratories, Wilmington, MA).

Techniques:

Journal: BMC Developmental Biology

Article Title: Endogenous expression of ASLV viral proteins in specific pathogen free chicken embryos: relevance for the developmental biology research field

doi: 10.1186/1471-213X-10-106

Figure Lengend Snippet: List of primers used in this study and target ASLV regions and subgroups

Article Snippet: Antibodies and dilutions used were as follows: AMV-3C2 mouse monoclonal antibody raised against the ASLV viral matrix protein p19 (1:5; NICHD-funded Developmental Studies Hybridoma Bank maintained by The University of Iowa, Iowa City, IA); p27 rabbit polyclonal antibody raised against the ASLV viral capsid protein p27 (1:1000; Charles River Laboratories, Wilmington, MA).

Techniques:

Journal: BMC Developmental Biology

Article Title: Endogenous expression of ASLV viral proteins in specific pathogen free chicken embryos: relevance for the developmental biology research field

doi: 10.1186/1471-213X-10-106

Figure Lengend Snippet: Immunohistochemical detection of endogenous viral proteins in SPF embryos

Article Snippet: Antibodies and dilutions used were as follows: AMV-3C2 mouse monoclonal antibody raised against the ASLV viral matrix protein p19 (1:5; NICHD-funded Developmental Studies Hybridoma Bank maintained by The University of Iowa, Iowa City, IA); p27 rabbit polyclonal antibody raised against the ASLV viral capsid protein p27 (1:1000; Charles River Laboratories, Wilmington, MA).

Techniques: Immunohistochemical staining