species to another may not be the same because of the differences and divergence of
CB2Rs across species (Ndong et al. 2011 ; Onaivi et al. 2015 ).
10.4 Genetic Manipulation of CB2 Cannabinoid
Receptors
Many previous studies could not detect the expression of CB2Rs in the brain
(Brown et al. 2002 ; Galiegue et al. 1995 ; Munro et al. 1993 ), because the poly-
merase chain reaction (PCR) primers may not have been specific to detect CB2R
isoforms. The specificity of the available antibodies for both CB1Rs and CB2Rs has
also been controversial as some could not detect the native and in some cases the
transfected cannabinoid CBR antigen, although they recognized proteins in Western
blot and in immunohistochemical analysis (Grimsey et al. 2008 ). There are also
problems with the antibodies because of the species differences between human and
rodent CB2R gene. We have resolved some of these issues by using CB2R isoform
specific TaqMan probes that could differentiate the isoform-specific expression and
are more sensitive and specific than CB2R antibodies that are currently available.
The controversial CB2R brain expression could also be due to the low expression
levels of CB2A isoform in brain regions and the less specific CB2R commercial
antibodies in immunohistochemical studies, especially in those studies using anti-
bodies against human hCB2 epitopes for rodent brain immunostaining. There are
also problems with the use of the CB2R knockout (KO) mice in Western blots and
in behavioral analysis (Buckley et al. 2000 ). When we analyzed the CB2R KO mice
using the three TaqMan probes against two promoters of mouse CB2R gene and the
deleted part of CB2R gene, we found that the promoters of CB2R KO mice were
still active and that a CB2R truncated version was expressed, indicating that the
CB2R KO mice with ablation of the C-terminal peptides of 131 amino acids
(Buckley et al. 2000 ) was an incomplete CB2R knockout. Another CB2R KO
mouse line that has now been generated with the ablation of N-terminal peptide 156
amino acid may clarify the specificity of the antibodies that were used against the
N-terminal epitopes. Comparison of these two CB2R mutant mice suggested that
genetic background and/or unknown effects on other signaling pathways may
contribute to the observed results obtained from the use of the currently available
CB2R mutant mice (Malfitano et al. 2014 ). Thus, contrary to prior reports that
CB2Rs were not functionally expressed in neurons, we and others have now
reported the wide distribution of CB2Rs in brain regions, suggesting a re-evaluation
of the role of CB2Rs in the CNS.
The complete gene structure, 5’- and 3’-UTR, and transcription initiation sites of
human CB2Rs have not been fully characterized (Abood 2005 ; Onaivi et al. 2006 ),
until now. After we and others identified and reported mouse CB2R expression in
brain regions (Gong et al. 2006 ; Van Sickle et al. 2005 ), the specific expression of
human or mouse CB2R isoforms in brain regions was not known. But the published
232 E.S. Onaivi et al.