Immortalization of common marmoset fibroblasts using piggyBac-mediated hTERT transgenesis

As part of this study I wanted to immortalize common marmoset fibroblast by robust long-term expression of human telomerase reverse transcriptase (hTERT) using the piggyBac trans-poson system. Human TERT immortalization circumvents cell senescence and crisis and al-lows virtually infinite cell proliferation. Such cells would allow long-term studies with fibro-blast-like cells without the potential severe effects of cell senescence likely to occur in prima-ry fibroblasts.

3.4.1 Exogenous hTERT prolongs cell proliferation potential in common marmoset fibroblasts

To immortalize common marmoset fibroblasts I used human telomerase reverse transcriptase (hTERT). For the hTERT transgene insertion into the fibroblast genome we used the piggyBac transposon system. The hTERT cDNA was cloned into the piggyBac transposon system as described in 2.4.5.

Fibroblasts were cotransfected (2.15.6.1) with the piggyBac plasmid containing the hTERT cDNA (3.8 A) and the PBase plasmid (Figure 3.2 C) catalyzing the integration of the piggy-Bac transposon system. After transfection, the transfected cells were puromycin selected for ten passages. The first generated cells were polyclonal (derived from different cells) and were named polyclonal hTERT cells. Additionally the polyclonal cells were clonally expanded as described in 2.15.4, to obtain monoclonal (derived from one cell) cell lines. Two monoclonal

Results 66

cell lines (#1, #4) were generated and further passaged as well as the polyclonal cell line. The cell lines were passaged in parallel to non-transfected primary fibroblasts (as control). To ex-amine whether the hTERT transfected cells have an extended ability to replicate, we calculat-ed the population-doubling level (PDL) (2.15.5) to get an estimate of their proliferation poten-tial. The PDL is an approximation of the occurring population doublings over time (passages).

The same was performed for the control cells. As expected, the hTERT transfected cells clear-ly show higher proliferation potential then primary fibroblasts (Figure 3.8 B). The hTERT transfected cells underwent several hundred population doublings until the experiments were terminated (Figure 3.8 B). In contrast, the primary fibroblast controls underwent a significant-ly

Results 67

Figure 3.8. A) piggyBac construct used for immortalization. Human TERT is under control of the CAG promoter (Pcag). pA: poly A signal; 5ˈ-TR: 5ˈ-terminal repeat; 3ˈ-TR: 3ˈ-terminal repeat;; P: puromycin resistance gene.

B) hTERT Cell Population-doubling level (PDL). PDL was determined by using cell counts and the respective number of passages. PDL was calculated with the formula: PDL(n/n-1) = log (Nf/N0)/log 2; n=passage number, Nf=final number of cells, N0 = number of cells seeded at passage.

lower number of population doublings, reaching only a PDL of 46 and 18, respectively (Fig-ure 3.8). These experiments were performed over a timeframe of more than nine months.

Phase contrast microscopy of the cells revealed a morphological change of the immortalized cells in comparison to proliferating fibroblasts. The immortalized cell lines exhibit a thin and

“needle like” shape (Figure 3.9 A). Such changes in morphology could indicate changes in gene expression, as common in cancer cells. Images of non-proliferating fibroblasts in a high passage show the typical morphology of cells in senescence (Blagosklonny, 2006). They show an enlargement of the cytoplasm (cell hypertrophy), with change from the fibroblast typical narrow, drawn-out morphology to a broader, rounder morphology (Figure 3.9 A). The

Results 68

immortalized cell lines still exhibit proliferation activity, even in high passages (Figure 3.9 A). To determine hTERT transgenesis, I performed immunofluorescence (IF) staining using a TERT specific antibody (2.17). Staining was performed using the hTERT immortalized cell lines as well as fibroblasts and ES cells as controls (2.17).

All three immortalized cell lines (polyclonal, #1, #4) showed expression of TERT (Figure 3.9 B). Additionally, it was observed that the hTERT immortalized cell lines have smaller sized nuclei than primary fibroblasts (Figure 3.9 B), further indicating changes associated with can-cerous transformation. Furthermore, we performed RT-PCR (2.2, 2.9) to show hTERT mRNA expression in the immortalized cell lines (Figure 3.9 C).

These results indicate cell immortalization in common marmoset fibroblasts through hTERT transgene expression. However, the generated cell lines do not show the same morphology as primary fibroblasts. To our knowledge, no hTERT immortalized common marmoset cell lines have been reported so far.

Results 69

Figure 3.9. A) Phase contrast images of the hTERT immortalized cell lines and fibroblast controls. P: passage.

Clone #4 and the polyclonal cell line show a thinner more “needle like” morphology when compared to fibro-blasts. Fibroblasts in P26 show a flat and broad morphology typical for cells in senescence. Bars = 200µm B) hTERT immunofluorescence staining of immortalized cell lines. Immortalized cell lines show expected TERT expression (red). No expression was detected in fibroblasts. Bars = 200µm. Only images of clone #4 are shown because clone #4 and #1 are indistinguishable from each other C) RT-PCR for expression of hTERT. Primer pairs specific for the hTERT transgene were used. As a loading control, β-Actin was amplified. As template DNA, cDNA of the immortalized cell lines and fibroblasts was used. Fibroblasts of two different common mar-mosets were used as control. For all templates –RT controls were used.

Discussion 70

4. Discussion

The aim of this study was to establish methods for the genomic modification of the common marmoset monkey. This would extend the spectrum of possible applications of the common marmoset as a model organism in biomedical and preclinical research. The different parts of this study were i) the generation of transgenic common marmosets carrying a pluripotency specific marker cassette ii) establishing CRISPR/Cas9 system, a new and promising method for gene targeting, in common marmoset monkey cells, and iii) generation of immortalized common marmoset fibroblast cell lines, and the generation of marmoset iPS cell lines by ro-bust expression of transgenes from the piggyBac transposon.