A transgene containing theOMP promoter, followed by a stop cassette (con-taining a CDS but no polyadenylation site), the CDS of the GFP marker and the polyadenylation signal of the rabbitβ-globin was constructed in the laboratory (Rossier, 2007). The stop cassette contained the diphteria toxin receptor CDS (DTR, also called heparin binding EGF-like factor proprotein, or proHB-EGF). This transgene was calledOMP-D-GFP. Three mouse lines (termed OMP-D-GFP#1, OMP-D-GFP#2 and OMP-D-GFP#3) were ob-tained.
Unexpectedly, all three transgenic lines showed detectable GFP expres-sion in the olfactory system before Cre recombination (Figure 6.14A-B on page 79, data shown only for two lines). According to expectations, Cre recombination led to an increase in the number of GFP-positive sensory neu-rons, and glomeruli (Figure 6.14D-E on page 79).
We observed a similar “leaky stop cassette” in an independent mouse line, based on the same strategy, but in which Channelrhodopsin-2 (ChR2) re-placed the GFP sequence. Given that ChR2 was fused with the YFP marker (OMP-D-ChR2 mouse line), ChR2 was easily visualized (Figure 6.14C,F on page 79).
A western blot performed on extracts from the threeOMP-D-GFP mouse lines before Cre recombination showed that the size of the expressed GFP protein was about 27 kDa (Figure 6.15 on page 80). This is the expected size of the GFP protein, and the one observed in an OMP-GFP line (a knock-in lknock-ine, Potter et al., 2001) (Figure 6.15 on page 80). The leakiness of the transgene is therefore likely the result of either a splice of part of the stop cassette or to the non-translation of the first ORF. But not of the expression of a fusion protein between the product of an ORF located upstream of the GFP CDS and GFP.
wt
A GFP
line #1
B
line #3 line #2
C YFP
ChR2::YFP pA OMP
GFP pA
OMP
D E F
OMPCre
OMP-D-GFP OMP-D-ChR2
Figure 6.14: Transgene-driven GFP expression in the olfactory system. (A-B) In mice carrying the OMP-D-GFP transgene, the GFP marker is expressed in the MOE (not shown) and in the OB prior to Cre recombination. (D-E) Following Cre recombination, an increase in the number of GFP-positive glomeruli is observed. (C,F) A similar line, in which the GFP cassette has been replaced by the coding sequence of the fusion protein ChR2::YFP, shows the same phenotype before and after Cre recombination. Modified from Rossier, 2007. OMP-D-ChR2 images were kindly provided by Matsuo Tomohiko.
Scale bar, 400µm.
6.3.2 Expression of V1rb2 in the Olfactory System
Despite the relatively leakiness exhibited by the OMP-D-GFP transgene, we generated a corresponding transgene with V1rb2 placed after the stop cassette.
Pronuclear injection of the OMP-D-V1rb2 resulted in a total of 75 pups and, among them, 14 bearing the transgene. Expression of the transgene was evauated by in situ hybridizations. Among 8 lines analysed, two lines
OMP-D-GFP (V) (B) (C)
#1 #2 #3
Figure 6.15: The GFP protein in OMP-D-GFP mouse lines. West-ern blot for the GFP protein. OMP-D-GFP transgenic lines show different level of expression of the GFP protein before removal of the stop cassette.
(V) =OMP-D-V1rb2, (B) = C57BL/6 and (C) =OMP-GFP. The band for the OMP-GFP line was obtained with a lower exposition time and then normalized to the other bands.
(OMP-D-V1rb2#7 andOMP-D-V1rb2#12) showed no transgene expression, but 6 of them exhibited very strong V1rb2 transcription in the MOE (Fig-ure 6.16A,E,I on page 81). This was in absence of the Cre recombinase, and represented the first time we were able to overexpress a V1rb2 transcript in olfactory sensory neurons. In the VNO, V1rb2 was overexpressed only in the basal layer, where generally V2Rs and M10/M1 MHC families are tran-scribed (Figure 6.16B,F,J on page 81). The presence of the V1RB2 protein itself could not be assessed directly because of a lack of antibody against this receptor (despite our very numerous attempts to generate such anti-body). Based on our observations with the OMP-D-GFP line, in which the expressed GFP protein was apparently unaltered before Cre recombination, we considered that the presence of V1rb2 transcripts in the OMP-D-V1rb2 line meant the presence of the V1RB2 protein.
We then removed the stop cassette by crossing the transgenic lines with OMP-Cre mice (Li et al., 2004), expecting an increase in the expression of the transgene, similarly to what we observed for the OMP-D-GFP lines.
Surprisingly, the removal of the stop cassette led to a complete silencing of the OMP-D-V1rb2 transgenes. This in all lines analysed, and in both the VNO and the MOE (Figure 6.16C-D,G-H,K-L on page 81).
All lines exhibited similar if not identical expression patterns. We kept 3 of them (OMP-D-V1rb2#2, OMP-D-V1rb2#4 and OMP-D-V1rb2#10), and all further experiments were performed on theOMP-D-V1rb2#2 line if
line
#10 line
#4 MOE
V1rb2 pA OMP
VNO
w t
OMPCre
w t
OMPCre
w t
OMPCre
A
C
E
line
#2
G
I
K
B
D
F
H
J
L
OMP-D-V1rb2
Figure 6.16: V1rb2 overexpression in the olfactory system in OMP-D-V1rb2 mouse lines. In situhybridizations of coronal sections of the head of mice belonging to 3 different OMP-D-V1rb2 mouse lines. Overexpression ofV1rb2 is clearly visible in both the MOE and the VNO in absence of the Cre recombinase. In the VNO, overexpression is limited to the basal layer, where V2Rs and M10/M1 MHC families are expressed (B,F,J). Strong punctate labelling in the VNO corresponds to the endogenous expression ofV1rb2. Scale bar, 200µm.
not indicated otherwise.