Etablierung der RCAS-Gentransfertechnologie zur Durchführung funktionell-genomischer Studien beim Haushuhn

Introduction of the RCAS retroviral gene transfer technology for
functional genomic studies in the chicken The recently sequenced
chicken genome now allows the identification of so far unknown
genes. Some of them may play a potential role in the regulation of
the immune system. In future studies roles of these genes have to
be analyzed. At this in vitro studies will be used firstly. But in
vivo functions of those genes will have to be described in the end.
Therefore techniques for a fast and efficient over expression and
knock down of candidate genes have to be established. Goal of this
work was to prove the applicability of the RCAS retroviral vector
system, which has been developed in the 1980s. This highly
efficient system is based on a replication competent Rous
Sarkoma-Virus and has already been used in cell and developmental
biology studies in the chicken. However studies on its application
after hatch haven´t been performed so far, despite its particular
relevance for studies on the chicken immune system as it is widely
immature at the time of hatch. In the first part of this work, a
green fluorescent protein (GFP) expressing vector was constructed.
An influence of the chicken line on the susceptibility of chicken
embryo fibroblasts was detected in flow cytometric analyses. A
possible reason could be the presence of endogenous retroviruses in
less susceptible chicken lines leading to receptor interference. In
accordance with this observation in in vivo tests retrovirus free
chickens showed more homogenous plasma titers of RCAS based protein
than a commercial chicken line. As GFP expression was predominantly
seen in endothelial cells, the RCAS technology can be considered as
particularly eligible for the expression of soluble proteins.
Additionally viral protein was demonstrated to be expressed in
bursal cells and heart muscle. For further characterization of the
RCAS system vectors for the expression of the cytokine chBAFF or a
soluble chBAFF receptor construct (huBCMA-Fc) were used. Plasma
expression of biologically active cytokine was detectable
throughout the entire experiment (two months).
RCAS(BP)A-chBAFF-Flag transduced birds showed in comparison to
controls significantly increased B cell frequencies in the spleen
as well as enhanced plasma antibody titers. On the other hand
animals expressing the soluble receptor huBCMA-Fc showed a
significant reduction of splenic B cell frequency and an inhibited
development of the bursa of fabricius. These findings correspond to
the data obtained in studies with recombinant proteins. In the
second part of this work the experiences with the RCAS technology
were used to evaluate the function of the CD40/CD40L system as a
second cytokine system. A soluble chCD40-huFc fusion protein was
constructed as a functional inhibitor for CD40/CD40L interaction.
In vitro tests showed that chCD40-huFc was able to bind a soluble
chCD40L fusion protein. In vivo the RCAS based expression of
chCD40-huFc caused a reduction of the blood B cell frequency and
the bursa weight. Possibly the functional inhibition of the
CD40/CD40L system decreases the number of B cells emigrating from
the bursal medulla. Unlike in humans with a defect in the CD40L
gene in chickens expressing chCD40-huFc the plasma IgM titer was
not increased. However plasma titers of IgA and IgG were decreased,
comparably with humans and mice with CD40L defects. The RCAS gene
transfer technology was proved in two different model systems as an
excellent system for analyses of cytokine functions in the chicken.
Furthermore this system could also be used for functional analyses
of growth factors and hormones.