KLINIKUM PART

The sera of the mice (expecially the one immunized with URE-10 peptide) show titer against FoxP recombinant protein. In september we can proceed with the hybridoma using the mouse immunized with ure-10

UNIVERSITY PART

Immunostaining against ELAV and BruchPilot in FoxP-IsoB-Gal4 flies (ELAV: 20x and 40x, BruchPilot 20x)

BruchPilot alone

Cell counting of 2 adult brains  for the Stinger staining in FoxP-IsoB-Gal4 flies. The expression is symmetrical and similar in the 2 brains. I am planning to do some more staining and counting at least 10 brains that I will compare with the FoxP all isoforms-Gal4

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

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

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

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

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

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.