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5.2.6.2.1. Testing Functionality of Transgenes: Expression in the Skin Transplanting HSC into compromised mice results in HSC expansion and differentiation, that does not occur in normal homeostasis conditions. In order to analyze effects of candidate genes on hematopoiesis under homeostasis conditions transgenic mice carrying inducible expression constructs of candidate genes were generated by Dr. Timm Schroeder in Japan. Expression of a candidate gene either as a fusion gene or from an IRES construct was driven by a chicken beta actin promoter (pCAG). Insertion of a loxP site flanked STOP cassette in front of the promoter rendered the expression cassette inactive. CRE expression in these cells results in recombination of the two loxP sites thereby excising the STOP cassette and initializing gene expression by the pCAG promoter. By crossing transgenic mice containing this expression cassette to mouse lines expressing CRE under cell and stage specific promoters the effects of transgene expression can be analyzed in a cell type and developmental stage specific manner during homeostasis.

The F1 offspring of the initial six transgenic founders were transferred to the Helmholtz center mouse facility. Mice were genotyped for VENUS presence in the genomic DNA and positive mice backcrossed to the C57/Bl6 background (Table 3). Genotype positive mice were further tested for functional VENUS expression upon CRE recombination. We decided to first use fibroblasts grown out of tail clips from transgenic mice to analyze for VENUS functionality. To this end, fibroblasts were isolated from the tails of transgenic mice and infected with a lentivirus expressing CRE and TOMATO fluorescent protein localized to the mitochondria. Using fibroblasts from our inducible mouse lines, successfully infected cells express TOMATO as well as CRE which then recombines the two loxP sites and excises the STOP cassette in front of the transgene, thus inducing expression of both the candidate gene and VENUS. Seven days after infection of the fibroblasts with the CRE virus cells were analyzed by FACS analysis to check for TOMATO and VENUS expression.

Analysis of infected and CRE recombined fibroblasts showed a large proportion of negative cells, that show high auto fluorescence in both TOMATO and VENUS channels (Figure 5.41A). Cells above the negative cells indicate TOMATO positive

Results 117 infected cells, whereas cells below the negative cells indicate VENUS positive cells. We did not see any VENUS positive cells in non-infected fibroblasts, therefore, we were sure that the latter cells were in fact double positive for TOMATO and VENUS, but appeared to be singly VENUS positive due to compensation artifacts.

In mice containing a Staufen1VENUS expression cassette we found mice from all three founders to be functional for VENUS expression in the skin (Table 2). Not all the mice of each line were found to be positive. This is probably due to the fact that from some mice only very few fibroblasts grew out of tail clips and therefore, we could have missed the low number of double positive cells. In mice containing the Staufeni IRES VENUSnucmem expression cassette we did not find fibroblasts from any mouse with functional VENUS expression. This means that Staufeni overexpression does not work in skin fibroblasts in these mice. In Pumilio2 IRES VENUSnucmem containing mice we found all four mice tested (1 Founder) to be positive for VENUS expression indicating functional Pumilio2 overexpression in these cells.

Table 5.2: Functionality of expression cassette in the skin of inducible transgenic mice. Functionality of

expression cassette was analyzed by FACS coexpression of VENUS and TOMATO in tailclip derived fibroblasts infected with CRE-IRES-mitoTOMATO.

Table 5.3: Inducible candidate gene expressing mice: breeding scheme and functionality of expression cassettes in bone marrow. Transgenic mice were bred to C57/Bl6 mice for

several generations as indicated. Functionality of expression cassette in transgenic mice was determined by coexpression of VENUS and TOMATO in CRE-IRES-mitoTOMATO lentivirus infected c-kit+_lin-

Results 119 5.2.6.2.2. Testing Functionality of Transgenes: Expression in Bone

Marrow Cells

As we were interested in the overexpression of candidate genes in the bone marrow, the presence of VENUS expression in skin fibroblasts is a good indicator for functionality in other tissues, but cannot substitute for a functional characterization of bone marrow cells themselves. We decided to analyze the bone marrow of all previously tested mice, including the ones that did not show expression in the skin. Therefore, we sorted c-kit+ _lin- _{bone marrow cells from the transgenic mice and} infected them with the same CRE expressing lentivirus we had used for the fibroblasts (Figure 5.41B). A functional inducible N-CadherinVENUS mouse and a C57/Bl6 mouse were used as positive and negative controls, respectively.

Re-analysis of cells was done after 6 days, when enough time had passed for infection, CRE expression and recombination and subsequent VENUS expression to have been completed. As shown in Figure 5.42C, cells were analyzed for TOMATO and VENUS expression. Gates were set using uninfected C57/Bl6 cells. Negative as well as positive control cells infected with mock control (IRES-mitoTOMATO) showed 22% TOMATO positive cells but no VENUS positive or double positive cells. This result shows that the infection per se worked and that the inducible system is not active without CRE recombination. C57/Bl6 cells infected with the CRE lentivirus showed again 22% of TOMATO positive cells but 0% of VENUS or double positive cells as these mice do not contain VENUS coding sequences in their genome. However, CRE lentivirus infection of cells from N-Cadherin VENUS mice resulted in 22% TOMATO positive cells as well as 3% VENUS and TOMATO double positive cells. With this system we could now test functionality of the transgenic mice in bone marrow cells.

Figure 5.41: Experimental setup testing VENUS functionality of transgenic mice. FACS analysis of infected tailclip derived fibroblasts of C57/Bl6 and candidate

inducible transgenic mouse. Red triangle: infected cells. Green ellipse: VENUS and TOMATO double positive cells (A). Sorting scheme of c-kit+_lin- _{bone marrow cells (B).}

FACS analysis of infected c-kit+_lin- _{from negative and positive control mice (C).}

Two to six mice from each of the six different founders were tested in the described manner from the following mouse lines: Staufen1VENUS, Staufeni_-IRES- VENUSnucmem, and Pumilio2-IRES-VENUSnucmem. Unfortunately we found that no progeny from the founders of either Staufen1VENUS or Staufeni_-IRES- VENUSnucmem mouse lines expressed VENUS after CRE-mediated recombination in bone marrow cells (Figure 5.42 and Table 3). However, bone marrow cells from Pumilio2-IRES-VENUSnucmem mice showed 0.2-1.1% VENUS positive cells upon CRE

Results 121 recombination (Figure 5.42), and it was therefore possible that these mice contained functional expression cassettes in bone marrow cells.

Figure 5.42: Functionality of expression cassette in the bone marrow of inducible transgenic mice. Functionality of expression cassette was

confirmed by coexpression of VENUS and TOMATO in c-kit+_lin- _{derived cells}

infected with a lentivirus containing CRE-IRES-mitoTOMATO. FACS analysis of VENUS and TOMATO expression in c-kit+_lin-_{derived cells. The shown graphs are}

5.2.6.3. Tie2 Controlled Homeostatic Pumilio2 Overexpression Allows