Immunohistochemical detection of transgene expression in the brain using small epitope tags.
BACKGROUND
In vivo overexpression of proteins is a powerful approach to study their biological function, generate disease models or evaluate gene therapy approaches. In order to investigate an exogenously expressed protein, specific and sensitive detection is essential. Unfortunately, antibodies that allow histological detection of the protein of interest are not always readily available. The use of an epitope tag fused to the protein can circumvent this problem as well as provide the possibility to discriminate endogenous from overexpressed proteins. In order to minimize impact on the bioactivity and biodistribution of the overexpressed protein, preference is given to small tags.


RESULTS
In the present study, we evaluated several small epitope tags together with corresponding anti-tag antibodies for the detection of overexpressed proteins in rat brain, using eGFP as a reference. We generated several lentiviral vectors encoding eGFP with different N-terminally fused small epitope tags (AU1, flag, 3flag, HA, myc and V5). After confirmation of their functionality in cell culture, we injected these lentiviral vectors stereotactically into the striatum of rats and prepared paraformaldehyde fixed floating sections for immunohistochemical analysis. Using multiple antibodies and antibody dilutions per epitope tag, we extensively assessed the efficiency of several anti-tag antibodies for chromogenic immunohistochemical detection of the epitope tagged eGFPs by determining the proportion of immunoreactivity detected by anti-tag antibodies compared to anti-GFP antibody. Using fluorescence immunohistochemistry and confocal microscopy, we also quantified the proportion of eGFP-positive cells detected by anti-tag antibodies. Our results show that all the examined small epitope tags could be detected by anti-tag antibodies both in cell extracts as well as in vivo, although to varying degrees depending on the tag and antibody used. Using the presented protocol, V5/anti-V5 and HA/HA11 tag/antibody combinations provided the most sensitive detection in brain tissue. We confirmed the applicability of these optimized in vivo tag detection conditions for a difficult to detect protein, firefly luciferase (fLuc), using lentiviral vector constructs expressing V5 tagged and 3flag tagged fLuc protein.


CONCLUSIONS
We show here that several small epitope tags are useful for immunohistochemical detection of exogenous proteins in vivo. Our study also provides a generic methodology which is broadly applicable for the detection of overexpressed transgenes in mammalian brain tissue.
Lobbestael
Evy
E
Laboratory for Neurobiology and Gene Therapy, Division of Molecular Medicine, Department of Molecular and Cellular Medicine, Katholieke Universiteit Leuven, Leuven, Belgium.
Reumers
Veerle
V
Ibrahimi
Abdelilah
A
Paesen
Kirsten
K
Thiry
Irina
I
Gijsbers
Rik
R
Van den Haute
Chris
C
Debyser
Zeger
Z
Baekelandt
Veerle
V
Taymans
Jean-Marc
JM
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001921:Brain / Q000378:metabolism*; D002460:Cell Line; D000939:Epitopes / Q000235:genetics* / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D019076:Transgenes
D000906:Antibodies; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins