The aims of this study were to determine expression pattern and subcellular localisation of Pelo protein, and generation and analysis of Pelo conditional knockout mice.
In order to study the expression of Pelo at the protein level, we have generated polyclonal anti-Pelo antibody that was raised against the Pelo-GST fusion protein. The affinity purified anti-Pelo antibody specifically detects a protein of 44-kDa in Western blot analysis, which is similar in length to the predicted molecular weight of Pelo protein. Western blot analyses revealed the presence of the 44-kDa Pelo protein in all studied cell lines and adult mouse tissues.
Immunofluorescence staining of mouse embryonic fibroblasts with the affinity purified anti-Pelo antibody revealed that the Pelo is localised to the cytoskeleton and perinuclear region. Coimmunofluorescence staining of fibroblasts with anti-Pelo antibody and phalloidin, which specifically binds to stress actin filament, revealed the association of Pelo to the stress actin filament. Localisation of Pelo to cytoskeleton was consistent with the localisation of GFP in transgenic GFP-Pelo fibroblasts. Western blot analysis with protein extracted from different cellular fractions of testis revealed the only presence of the 44-kDa Pelo protein in cytoskeleton and membrane fractions. The absence of Pelo in nuclear fraction suggests that Pelo is localised in cytoskeleton and perinuclear region.
To investigate the consequence of Pelo knockdown on cell proliferation, Tet inducible siRNAs system was performed. Three siRNA sequences targeting different regions within the Pelo mRNA were tested for their efficiency to specifically knockdown Pelo in cell culture. The Western blot and Northern blot analyses revealed the failure of the three Pelo-siRNAs to RNAi-mediated gene silencing of Pelo.
To circumvent early embryonic lethality of conventional Pelo null mice being able to investigate the in vivo roles of murine Pelo gene during later stages of embryonic development and adult, we generated Pelo conditional knockout mice using inducible Cre/loxP recombination system. We successfully generated several high chimeric male mice, which transmitted the Peloflox allele to their offspring. The Peloflox/+ mice were then intercrossed with Peloflox/+or with Pelo+/-mice to generate Peloflox/floxandPelo flox/-animals in F2 generation. All Peloflox/flox and Peloflox/- mice are viable and fertile
indicating that the insertion of loxP and neomycin cassette (Neo) in intron 1 and 3’
flanking region of the gene did not disrupt the Pelo allele.
To determine whether the floxed Pelo allele can be deleted in vivo, Pelo floxed mice were mated with EIIaCre transgenic mice in which the adenovirus EIIa promoter directed the action of Cre recombinase gene to the single-cell zygote. The complete excision of the floxed Pelo (Peloflox) allele in Peloflox/+EIIaCre double transgenic mice clearly showed that the generated Pelofloxallele can successfully recombine with Cre recombinase in vivo system. As expected, we did not find Pelo¨/-EIIaCre mice in F2 generation suggesting that these mice died during embryonic development like the conventionalPelodeficient mice.
To achieve temporal inactivation of floxed Pelo allele in vivo and to study distinct function of Pelo gene, Cre-ERT fusion allele was introduced in the genome of Pelo flox/-mice. Tamoxifen (TAM)-treated Peloflox/-CreERT mice are subfertile. Histological analysis of testes from TAM-treated Peloflox/-CreERT mice revealed severe germ cell depletion. The increase of the germ cell depletion in testes of TAM-treated mice suggests that Pelo is essential for spermatogenesis.
Moreover, Southern blot analysis of different tissues of TAM-treated Peloflox/-CreERT mice revealed that the efficiency of Peloflox deletion was high in spleen (<50%) and less efficient in kidney and tail (~25%). Western blot analysis revealed that the significant decrease of protein level of Pelo in testes of TAM-treated Peloflox/-CreERTmice with severe germ cell depletion, while that with less germ cell depletion did not show a significant downregulation of the Pelo in testis. These results revealed the inducible deletion of floxed Pelo allele by CreERTdid not occur in all testicular cells.
To study the consequences of Pelo deficiency on cell proliferation, mouse embryonic fibroblasts (MEFs) were isolated from Peloflox/-CreERT and Pelo+/+CreERT embryos.
Southern blot anaylsis showed that the Cre-mediated recombination occurred partially in OHT-treated fibroblasts of Peloflox/-CreERTmice. The proliferation assay of OHT-treated Peloflox/-CreERT fibroblasts suggests that either Pelo deficiency does not influence cell proliferation or that the Pelo¨/-CreERTcells might be rapidly diluted in culture by fast growth of the nondeleted cells (Peloflox/-CreERT). Karyotype analysis of OHT-treated MEFs revealed an increased rate of aneuploid cells in analysed metaphases
high expression of transgenic allele only in testis of one transgenic line. In the other transgenic line, expression of transgenic allele was detectable in all studied tissues.
The second transgenic mice (hUB-Pelo) contain the human Pelota cDNA under the control of human ubiquitin C(hUbC) promoter. The expression analysis revealed that the expression of the transgenic transcript is restricted in testis of three generated lines.