Bmper-GCE Allele Characterization
Authors: Jinjin Guo, Jill McMahon, M. Todd Valerius, and Andrew P. McMahon
Submitted: 25 April 2011
Version: 3 - Final
Findings: Not VALIDATED
Expression of Bmper-CreERT2 was expected in the condensed nephrogenic
mesenchyme; including the comma and S-shaped bodies and potentially in the
developing vasculature of the kidney. BmperGCE/+; R26RlacZ/+ embryos were examined
between E15.5-16.5 dpc for GFP fluorescence and Tamoxifen induced ß-galactosidase
activity. No activity was detected in the expected cell populations.
Data:
Crosses
The Bmper-GCE strain is a BAC transgenic line with eGFPCreERT2 (GCE) expressed in
the BMP-binding endothelial regulator domain. Pronuclear injection of BAC DNA into
B6/DBA F1 embryos resulted in the birth of 60 pups of which 11 males and 11 females
carried the transgene. Of the 9 founders tested 8 transmitted the transgene but no GFP
fluorescence was detected in embryos from 15.5dpc-16.5dpc (Table 1).
Construct
Bmper-GCE
Date of
Birth
5-Sept-10
Pups
born
60
Founders
11M, 11F
Founders
mated
M5
M11
M13
M18
M36
M38
M48
M50
M60
Transmittal
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Visible
Reporter
No
No
No
No
No
No
No
nd
No
Antibody
to
reporter
nd
nd
nd
nd
nd
nd
nd
nd
nd
Table 1: Summary of founder analysis for GFP reporter activity.
Genotyping
Tail samples of the embryos were collected and incubated in tail digestion buffer
overnight at 55ºC. PCR was performed as per the protocol below and the PCR products
were run on a 1.5% agarose gel (Fig1).
Oligonucleotides: for targeted/transgenic allele Size: ~391bp
DNA sequence (forward): 5’- TAACCCGCTGAGAGCAACTC -3’
DNA sequence (reverse 2) 5’- GAACTTCAGGGTCAGCTTGC -3’
Amplifies 5’ arm into GFP sequence within GFP-Cre region.
Rxn Buffer and Conditions: (25µl reaction)
10X Gitschier Buffer (GSB):
670 mMTris, pH 8.8
166
Ammonium
Page
1 ofmM
5, printed
2/9/16. Sulfate
65 mM MgCl2
0.1% gelatin
10X GSB
25mM dNTP
10uM primer F
10uM primer R
DMSO
2.5ul
1ul
1ul
1ul
2.5ul
2-mercaptoethanol
Amplify Taq
5x cresol red dye
Genomic DNA
Total volume
0.125ul
0.2ul (5u/ul)
5ul
1ul
25 ul
94ºC
94ºC
55.5ºC
72ºC
72ºC
3min 1 cycle
30sec
30sec 35cycles
45sec
10min 1 cycle
Fig1: Numbers 48, 52, 54, 55, 58, 59, 61 BmperGCE/+, P: BmperGCE/+ positive control, W:
Wildtype control, N: Negative control.
Cre-recombinase Activity
The male founders were crossed with Rosa26RlacZ/+(R26R) mice to obtain BmperGCE/+;
R26RlacZ/+ embryos. In order to activate β-gal reporter expression from the
R26RlacZ/+allele, an intraperitoneal injection of Tamoxifen in corn oil was given to
pregnant mice at 2mg/40g body weight or 2 doses at 2mg/40g body weight. Tamoxifen
treated BmperGCE/+; R26RlacZ/+ embryos were dissected between 2-4 days post injection.
UGS samples were processed for X-gal staining but only low levels of ß-galactosidase
activity was detected in areas not consistant with the expected expression in either
nephrogenic mesenchyme or vascular progenitor cells (Table 2 and Fig 2, 3)).
Founders
mated
M5
Transmittal
Yes
ß-gal activity Tam (1X dose) ß-gal activity Tam (2X dose)
12.5-13.5
11.5-14.5
No
No
Correct Cre Activity
No
Page 2 of 5, printed 2/9/16.
M11
Yes
nd
No
No
M13
M18
M38
M48
M60
Yes
Yes
Yes
Yes
Yes
No
nd
No
No
No
nd
No
nd
nd
No
No
No
No
No
No
Table 2. Summary of founder analysis for Cre-dependant ß-gal activity in
Tamoxifen induced BmperGCE/+; R26RlacZ/+ embryos.
Fig 2. Cre-dependant ß-gal activity in female BmperGCE/+; R26RlacZ/+ UGS.Two doses
of 2mg/40g Tamoxifen, injected at 11.5 and 13.5dpc resulted in weak ß-gal activity on
the surface of the kidney and reproductive tubes but not as expected in condensed
nephrogenic mesenchymal derivatives.
Page 3 of 5, printed 2/9/16.
Fig 3. Cre-dependant ß-gal activity is not detected in the cortical region of
BmperGCE/+; R26RlacZ/+ UGS.Two doses of 2mg/40g Tamoxifen, injected at 11.5 and
13.5dpc did not resulted in detectable ß-gal activity in condensed nephrogenic
mesenchyme.
Immunohistochemistry
Immunohistochemistry was performed to examine if the eGFPCreERT2 allele was
expressed in the expected Bmper domain. To test for Cre function, BmperGCE/+;
R26RlacZ/+ UGSs from Tamoxifen injected mice were assayed. β-gal and GFP
expression were examined by probing with rabbit anti-β-gal, chicken-anti-GFP and antirat-PECAM CD31 antibody.
Whole UGSs were fixed in 4% paraformaldehyde at 4ºC 2 hours, washed 3 times in
PBS, equilibrated in 30% sucrose overnight and then embedded in OCT and flash
frozen on dry-ice. The UGSs were sectioned at 16um and probed with rabbit-anti-ßgal/Chicken-anti-GFP/rat-anti-PECAM CD31. ß-gal (Rabbit, MP Biomedicals, LLC,
55976, 1: 20000), GFP (Chicken, Aves Labs, Inc, GFP-1020, 1:500) anti-PECAM CD31
(Rat, BD Pharmingen, 553370, 1:1000) were incubated overnight at 4ºC and detected
with secondary antibodies Alexafluor 488, 555, and 633 (Molecular probes).
Neither GFP nor ß-gal positive cells were detected in the cortical region of BmperGCE/+;
R26RlacZ/+ embryos at 15.5dpc (Fig 4).
Page 4 of 5, printed 2/9/16.
Fig 4. Neither GFP nor ß-gal positive cells are detected in condensed nephrogenic
mesenchyme in BmperGCE/+; R26RlacZ/+ UGS at 15.5dpc.
Page 5 of 5, printed 2/9/16.