Abstract
BackgroundThe use of Specific Pathogen Free (SPF) eggs in combination with RCAS retrovirus, a member of the Avian Sarcoma-Leukosis Virus (ASLV) family, is of standard practice to study gene function and development. SPF eggs are certified free of infection by specific pathogen viruses of either exogenous or endogenous origin, including those belonging to the ASLV family. Based on this, SPF embryos are considered to be free of ASLV viral protein expression, and consequently in developmental research studies RCAS infected cells are routinely identified by immunohistochemistry against the ASLV viral proteins p19 and p27. Contrary to this generally accepted notion, observations in our laboratory suggested that certified SPF chicken embryos may endogenously express ASLV viral proteins p19 and p27. Since these observations may have significant implications for the developmental research field we further investigated this possibility.ResultsWe demonstrate that certified SPF chicken embryos have transcriptionally active endogenous ASLV loci (ev loci) capable of expressing ASLV viral proteins, such as p19 and p27, even when those loci are not capable of producing viral particles. We also show that the extent of viral protein expression in embryonic tissues varies not only among flocks but also between embryos of the same flock. In addition, our genetic screening revealed significant heterogeneity in ev loci composition even among embryos of the same flock.ConclusionsThese observations have critical implications for the developmental biology research field, since they strongly suggest that the current standard methodology used in experimental studies using the chick embryo and RCAS vectors may lead to inaccurate interpretation of results. Retrospectively, our observations suggest that studies in which infected cells have been identified simply by pan-ASLV viral protein expression may need to be considered with caution. For future studies, they point to a need for careful selection and screening of the chick SPF lines to be used in combination with RCAS constructs, as well as the methodology utilized for qualitative analysis of experimental results. A series of practical guidelines to ensure research quality animals and accuracy of the interpretation of results is recommended and discussed.
Highlights
The use of Specific Pathogen Free (SPF) eggs in combination with RCAS retrovirus, a member of the Avian Sarcoma-Leukosis Virus (ASLV) family, is of standard practice to study gene function and development
The RCAS vectors (Replication Competent ALV LTR with a Splice acceptor) are laboratory-derived vectors engineered from the Rous Sarcoma Virus (RSV), a naturallyderived retrovirus originated from the Avian Leukosis Virus (ALV) [3]
Members of the Avian Sarcoma-Leukosis Virus (ASLV) family, which include the ALVs, RSVs and RCAS viruses, are classified into 6 subgroups (A-E and J) based on antigenic characteristics of their envelope glycoproteins. They are classified as being either exogenous or endogenous; ASLV subgroups A-D and J are “exogenous”: naturally occurring viruses that can be transmitted either vertically from dam to progeny through the egg, or horizontally from bird to bird, while members of the ASLV subgroup E are “endogenous” viruses: copies of exogenous retroviruses that have been integrated into the host germ line cells and are transmitted genetically in a Mendelian fashion [4]
Summary
The use of Specific Pathogen Free (SPF) eggs in combination with RCAS retrovirus, a member of the Avian Sarcoma-Leukosis Virus (ASLV) family, is of standard practice to study gene function and development. Members of the Avian Sarcoma-Leukosis Virus (ASLV) family, which include the ALVs, RSVs and RCAS viruses, are classified into 6 subgroups (A-E and J) based on antigenic characteristics of their envelope (env) glycoproteins They are classified as being either exogenous or endogenous; ASLV subgroups A-D and J are “exogenous”: naturally occurring viruses that can be transmitted either vertically from dam to progeny through the egg, or horizontally from bird to bird, while members of the ASLV subgroup E are “endogenous” viruses: copies of exogenous retroviruses that have been integrated into the host germ line cells and are transmitted genetically in a Mendelian fashion [4]. All members of the ASLV family share highly similar sequences for all the viral proteins except for the region encoding the subgroup specific gp surface envelope glycoprotein, which determines the ASLV subgroup classification and subgroup infectionspecificity [4]
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