FoxPE-Gal4 + REPO and BruchPilot
Stinger + REPO (20x and 40x): Visualization of gliacells (anti-Repo) and GFP-FoxPE expressing Neurons at 20x and 40x
CD8-GFP + BruchPilot
BruchPilot alone
PCR Amplification of HomI and HomII: Amplification of the Inserts for the Knock-Out of FoxPE
ELISA titer not changing
KLINIKUM PART
The ELISA titer for the FoxP antibody production is not changing with the number of the immunizations (on the contrary it looks slightly reduced). The titer of the peptide URE-10 looks better then the URE-9. We will proced with a fifth immunization.
Here the complete graph for URE-10 with the pre-bloood and the first immunization
CD8-GFP x FoxP-IsoB_Gal4
Not confocal immages (going to the confocal this week)
The construct I am analyzing is the FoxP-IsoB_Gal4, so with the Gal4 inserted in the 8th exon
Larvae
Adult VNC
The Gal4 line is working
Here some preliminary pictures (not confocal) of the FoxP.IsoB-Gal4 line crossed with a Stinger line (GFP expression only in the nucleus)
and with a 6xCD8-GFP (a little bit massacred), this week I will do them again
And here´s the whole larva for the 6xCD8-GFP
I will try to go to the confocal as soon as possible
Schemes for FoxP and FoxP isoB constructs
This is the technique we are using to create the non conditional constructs: HDR using CRISPR/Cas9 (S. J. Gratz et al., 2014)
and these are the schemes of the constructs we are creating
First KO construct ready + WB
UNIVERSITY PART
The first construct for the KO (ds-Red-attP plasmid) of FoxP (all isoforms) is ready, with both the homology domain (Hom1 and Hom2) incorporated in the plasmid
As for the constructs, we miss just the KO for FoxP IsoB (I will let the student do it).
I also made the Western Blot for the FoxP-GFP strain. The green bands are actin (used as internal control), while the GFP should be in red. The blot has some background, and even though we have a slight red band of the appropriate dimension I am not sure it is specific. So I will do the Western blot again using a FLAG antibody.
Serum detects recombinant FoxP IsoB
KLINIKUM PART
The serum of the mouse immunized with one of the peptides recognize recombinant FoxP IsoB (ELISA test)
Progresses in antibody production
KLINIKUM PART
After 2 immunization on the mice we made an ELISA test in order to see if there is an immunogenic response in the serum. There is a response for both the peptides, the only problem is that is not really specific: the mice immunized with one peptide (URE-9) show a response also for the other peptide (URE-10), even if slightly lower, and viceversa.
Also the response is not great enough, therefore a 3rd immunization is required.
We also finally have the recombinant protein (FoxP IsoB-FLAG). Here´s the picture of the western blot with the band of the appropriate size (60 kD) on the right. On the left is the same band from the cell lysate.
