Top PDF Transgenic mice for a tamoxifen-induced, conditional expression of the Cre recombinase in osteoclasts.

We next investigated the specificity of recombination in embryos. CtsKCreERT2LacZ positive females were crossed with R26R males. Pregnant females were fed with tamoxifen for 3 days at day E13.5 of embryogenesis. Embryos were collected at day E17.5 and then processed to detect b-galactosidase activity in tissues. Embryos from CtsKCreERT2 lines #4 and #284 showed intense staining in long bones. Staining could also be seen in vertebrae, calvaria and craniums (Fig. 3). As expected, no staining was observed in CtsKCreERT22/2LacZ+/+ or C57BL/6 wild-type embryos. These results strongly suggest that the tissue-specific activation of the Cre recombinase had occurred in mouse line #4 and #284. To observe in more details the specificity of Cre recombination, paraffin sections of the b-galactosidase-stained embryos were prepared. Sections of strain #4 embryos showed an intense b-galactosidase activity in long bones (Fig. 4B; b.1, b.2 and b.4,), vertebrae (Fig. 4B; b.1 and b.6), ribs (Fig. 4B; b.2 and b.6), cranium and calvaria (Fig. 4B; b1). In contrast, CtsKCreERT22/2LacZ+/2 and C57BL/6 wild-type embryos did not exhibit any b-galactosidase activity in any tissue including bones (Fig. 4A and C), with the exception of the intestine as usually observed in all embryos. Sections of strain #284 embryos showed similar results (data not shown). It is worth noting that some b-galactosidase staining was observed at junctions between femur and tibia of embryos (Fig. 4B, b.4, b.5) as well as in the periostium (Fig. 4B, b.3; Fig. 5B, b.3). These mononucleated cells were not positive for the osteoclast marker TRAcP (Fig. 5B; b.3 left). Because joints, bone and its periostium are high vascularized, these cells could represent mononucleated osteoclast precursors already commit- ted to osteoclastic differentiation [16]. At higher magnifications,

Previous fate-mapping studies have shown that Cre-dependent recombination driven by GFAP or PLP promoters during embryonic development labels distinct glial and neuronal cell populations [2–4,18]. Here, we asked whether both promoters are active in separate lineages or whether coincident activity can highlight a distinct neural cell type or cellular subpopulation. For that purpose, we analysed the Cre-complementation of the GFAP- NCre and PLP-CCre transgenes in ROSY reporter mice (Fig. 2). We could identify Cre-induced EYFP expression in various brain regions. Co-immunostaining for the reporter EYFP and the astroglia marker protein S100b together with distinct morphology revealed that most of the cells in which recombination had taken place were bona fide astrocytes (Fig. 2). Interestingly, the recombination pattern suggested that cerebellar Bergmann glial cells as well as a glial population at the juxtaventricular zone were derived from progenitors with simultaneous GFAP- and PLP-gene activity (Fig. 2A–F). We counted the fraction of EYFP-labeled Bergmann glial cells as a percentage of all S100b-positive cells in the Purkinje cell layer of the cerebellum and found 2568% labeled cells (mean6sd; n = 4 mice; 400 cells counted in total). In contrast, cortex (Fig. 2J–L), hippocampus, striatum or cerebellar white matter (Fig. S6) showed only a few labeled cells. Interestingly, another radial glial cell type, the Mu¨ller cells of the retina, exhibited a similarly high recombination frequency as the Bergmann glia (Fig. 2G–I). Although labeled cells were mainly identified as astrocytes, we also detected throughout the brain of all transgenic crossings cells, which were identified as NG2 glia due to their expression of the chondroitin sulphate proteoglycan NG2 [19–21] (Fig. 2M–O).

since the Cre-loxP technology was first applied to conditionally express transgenes in mice in 1992 [1,2]. In recent years, the use of loxP-containing transgenes in transgenic mice has increased but the possibility of unintended effects of this approach has not been taken into consideration [5,7,8,9,10,11,12,13,14,37,38,39,40,41]. Our data demonstrate that, contrary to the widely held assumption that transgenes are integrated into the genome in a head-to-tail tandem array [5,7,8,9,10,11,12,13,14,28,29,30, 37,38,39,40,41], inverted transgene integration is common when multiple copies of the transgene are integrated into the genome. Therefore, the non-specific effects of the inverted loxP sites have been overlooked and can provide alternative interpretations to the previous studies using this conditional transgene expression strategy. For example, a mutant SOD1 transgene that was flanked by loxP sites was used to construct a model for amyotrophic lateral sclerosis (ALS), which was crossed with a P0-Cre driver line. It was intended to delete the mutant SOD1 gene from the Schwann cells, thereby diminishing the expression of the mutant SOD1 in this cell type. The investigators observed that the ALS phenotype was exacerbated and concluded that the SOD1 enzyme activity carried by the mutant protein was protective to motor neurons and the reduction of this activity was responsible for the observed result [10]. Based on our result, an alternative interpretation is that ablation of proliferating Schwann cells during development or during axonal degeneration caused the observed phenotype. In another case, the Cre-loxP strategy was used to conditionally express shRNAs that silenced the fibroblast growth factor receptor 2 (Fgfr2) gene. When the shRNA was ubiquitously induced by EIIa-Cre driver, embryonic lethality was observed. When the shRNA was induced in the limb bud of the mouse embryos, an enhanced cell death and malformed limbs developed. The authors attributed these effects to specific silencing of the Fgfr2 gene [7]. Based on our data, an alternative interpretation is that the non- specific effects associated with the Cre-loxP approach caused the observed phenotypes. In conclusion, it is imperative that investigators who use transgenic lines constructed with loxP- containing transgenes take the non-specific effect into consider- ation in the experimental design and the interpretation of the results.

Bmi1 is a member of the polycomb repressive complex 1 and plays different roles during embryonic development, depending on the developmental context. Bmi1 over expression is observed in many types of cancer, including tumors of astroglial and neural origin. Although genetic depletion of Bmi1 has been described to result in tumor inhibitory effects partly through INK4A/Arf mediated senescence and apoptosis and also through INK4A/Arf independent effects, it has not been proven that Bmi1 can be causally involved in the formation of these tumors. To see whether this is the case, we developed two conditional Bmi1 transgenic models that were crossed with GFAP-Cre mice to activate transgenic expression in neural and glial lineages. We show here that these mice generate intermediate and anterior lobe pituitary tumors that are positive for ACTH and beta-endorphin. Combined transgenic expression of Bmi1 together with conditional loss of Rb resulted in pituitary tumors but was insufficient to induce medulloblastoma therefore indicating that the oncogenic function of Bmi1 depends on regulation of p16 INK4A /Rb rather than on regulation of p19 ARF /p53. Human pituitary adenomas show Bmi1 overexpression in over 50% of the cases, which indicates that Bmi1 could be causally involved in formation of these tumors similarly as in our mouse model.

; Col2a1-Cre mutants. In addition, in growth plates of R26 floxneoWnt4 ; Col2a1-Cre mutants, the zone of Col2a1 hybridization was significantly smaller yet the Ihh zone essentially unchanged, indicating a decrease in proliferating chondrocytes. The decrease of the zone of proliferating chondrocytes may result from lower rate of proliferation in mutants or a higher percentage of cells exiting the proliferation state. However, an in vitro micromass culture assay has shown that infection by a retroviral-delivered Wnt4 did not decrease the rate of cell proliferation [18]. In addition, no detectable change of cell proliferation was observed by Wnt5a and 5b misexpresssion using the same system. Indeed, recent studies in transgenic mice demonstrated that Wnt5b could promote proliferation of chondrocytes in vivo [11]. These distinct results may reflect the difference between mouse and chick models, or differences between the in vitro and in vivo approaches. The ability of Wnt4 to decrease the zone of proliferating chondrocytes may represent an enhancement of the endogenous activity of Wnt4, a competition with the activity of Wnt5b that promotes proliferation of chondrocytes, or a mimicking of Wnt5a function that inhibits the transition from resting chondrocytes to pro- liferating chondrocytes. Moreover, since Wnt4 may accelerate the differentiation of chondrocytes, proliferating chondrocytes with overexpression of Wnt4 may exit the cell cycle rapidly, leading to narrower zone of proliferating chondrocytes.

Because Pim1 is regarded as a ‘‘weak’’ oncogene, we decided to study mPIN solely induced by Pim1 overexpression and also the effect of Pim overexpression in the physiological settings of hormone- induced mPIN and loss of one Pten allele. A summary of the mouse line genotypes used in this study are as follows: tgPim1 animals. ND: Not detected. Human PIM1 was not detected in WT nor PTEN-Het mice. D) Level of PIM1 protein in the mice of different genotypes measured by Western blot. The prostate of 2 animals (1 and 2) per cohort was processed to extract total proteins and run in a PAGE to allow further identification by western blot. Transgenic PIM1 was identified by myc-tag present in the transgene. Total PIM1 content was identified with a PIM1 antibody cross-reacting with human and mouse species. Activity was measured as Bad phosphorylation levels at S112. E) Representative pictures of high grade mPIN lesions developed after hormone treatment. To determine the development of mPIN lesions due to hormone treatment, 8-week-old untreated and hormone-treated (for 1 or 2 rounds) mice of each genotype were sacrificed and prostate tissue was obtained (see text for details). H&E staining of prostate tissue was used for mPIN grading. Pictures represent: No treatment: wt) pl-grade 0; tgPim1) pl-grade 1; PTEN-Het) pl-grade 5; tgPim1/PTEN-Het) pl-grade 8; 1 round of hormone treatment: wt) pl-grade 2; tgPim1) pl-grade 4; PTEN-Het) pl-grade 10; tgPim1/PTEN-Het) pl-grade 11; 2 rounds of hormone treatment: wt) pl-grade 6; tgPim1) p-grade 13.

The Cre/loxP system is a powerful tool for generating conditional genomic recombination and is often used to examine the mechanistic role of specific genes in tumorigenesis. However, Cre toxicity due to its non-specific endonuclease activity has been a concern. Here, we report that tamoxifen-mediated Cre activation in vivo induced the regression of primary lymphomas in p532/2 mice. Our findings illustrate that Cre activation alone can induce the regression of established tumors.

One type of Cre toxicity involves the action of Cre recombinase on cryptic pseudo-loxP sites, which are present in the endogenous mouse genome [54,55] but, to have any detectable effect, this may require prolonged expression of high levels of Cre recombinase [56,57,58]. Such action may result in mutations and chromosomal abnormalities, by introducing single-stranded and double-stranded DNA breaks even if Cre-mediated recombination does not occur [55,59]. This has been implicated as the cause of sterility in mice expressing Cre recombinase in spermatids [60]. In somatic cells a common phenotypic effect is likely to be reduced cell proliferation and cell cycle arrest [57,61] leading to increased cell death. This may be relatively well tolerated and it has been suggested that many Cre-expressing transgenic strains that appear superficially normal are not actually completely normal, as often the effects of Cre cytotoxicity would be ameliorated by a combination of developmental selection and adaptation [48]. Nevertheless, abnormal phenotypes have been reported as a result of Cre

CXCL12 has been proposed to induce JAK/STAT tyrosine phosphorylation and to promote their association with CXCR4, and small molecule JAK inhibitors reduced CXCR4 function in vitro [27, 29–31]. In turn, shortly after engaging CXCL12, CXCR4 signaling through JAK2 and JAK3, and several STATs, also promoted SOCS3 expression in IM-9 cell line [27, 32]. Since these findings were reported several studies examined the interplay between SOCS3, JAKs and STATs in CXCR4 signaling and downstream biological activities. Increased SOCS3 expression by genetic manipulation or by treatment with cytokines, was found to repress CXCR4-me- diated chemotaxis to CXCL12 in vitro, and to mobilize hematopoietic progenitors from BM [27, 33]. Conversely, conditional SOCS3 deficiency using the MMTV-cre approach, which pro- motes cre recombinase expression and recombination in some epithelial cells and in several hematopoietic cells including B-lineage cells, led to a significant accumulation of immature B cells in BM. It was proposed that SOCS3 negatively regulates FAK phosphorylation and integ- rin-mediated adhesion to the extracellular matrix [20]. However, other studies using primary cells and cell lines derived from JAK3-deficient patients, failed to detect any requirement for JAK2 and JAK3 signaling in CXCL12-mediated biological activities [34]. Furthermore, in mice conditionally deficient in SOCS3 specifically in B-lineage cells (using the same Igα promoter driving cre recombinase expression, Mb1 Cre/+ ) it was observed that developing B cell subsets were normally represented in BM and there was no evidence for an accumulation of late stage immature B cell subsets [21].

different teams tried to develop strategies in which the expression of a mutation would be rendered con- ditional (for a review, see Gu et al. 1994, Cohen- Tannoudji and Babinet 1998). These strategies are based on the remarkable properties of the Cre/loxP system. The first step (see Fig. 5) consists in cre- ating mice carrying alleles in which two loxP sites surround an essential part of the gene to be studied, without disrupting its activity, by placing them for example in the introns (we subsequently call these alleles ‘‘floxed’’ alleles). In the case of a gene whose null mutations are letal in the homozygous state, it is necesssary to verify that the mice homozygous for the floxed allele are viable. These mice are then crossed with a transgenic mouse expressing the Cre recombinase in a particular cell type, using appro- priate regulatory sequences in the transgene (the Cre recombinase promotes the deletion of the sequences located between the loxP sites and induces a null mutation in the cell type in which the transgene is expressed). This strategy is powerful since it not only avoids the embryonic lethality produced when all the embryo cells carry the mutation, but also al- lows to address the effect of this mutation in any tissue, so long as a line of transgenic mice express- ing protein Cre in the tissue concerned is available (Table IV, Tsien et al. 1996, Shibata et al. 1997, Harada et al. 1999, Kulkarni et al. 1999, Xu et al. 1999).

MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression post-transcriptionally. MiRNAs are implicated in various biological processes associated with obesity, including adipocyte differentiation and lipid metabolism. We used a neuronal- specific inhibition of miRNA maturation in adult mice to study the consequences of miRNA loss on obesity development. Camk2a-CreERT2 (Cre + ) and floxed Dicer (Dicer lox/lox ) mice were crossed to generate tamoxifen-inducible conditional Dicer knockouts (cKO). Vehicle- and/or tamoxifen-injected Cre + ;Dicer lox/lox and Cre + ;Dicer +/+ served as controls. Four co- horts were used to a) measure body composition, b) follow food intake and body weight dy- namics, c) evaluate basal metabolism and effects of food deprivation, and d) assess the brain transcriptome consequences of miRNA loss. cKO mice developed severe obesity and gained 18 g extra weight over the 5 weeks following tamoxifen injection, mainly due to in- creased fat mass. This phenotype was highly reproducible and observed in all 38 cKO mice recorded and in none of the controls, excluding possible effects of tamoxifen or the non-in- duced transgene. Development of obesity was concomitant with hyperphagia, increased food efficiency, and decreased activity. Surprisingly, after reaching maximum body weight, obese cKO mice spontaneously started losing weight as rapidly as it was gained. Weight loss was accompanied by lowered O 2 -consumption and respiratory-exchange ratio. Brain

detected; however, a very pronounced decrease in the frequency of MEPPs was also observed. This decrease in MEPP frequency is not the result of alterations in the readily releasable pool of vesicles. It also seems unlikely that the alteration in MEPP frequency is the result of de- creased exocytosis, endocytosis, and total pool of ves- icles, as FM1-43 experiments have shown no difference in these parameters between wild-type and VAChT KD HOM mice. We hypothesized that, if in synapses, the number of copies of VAChT per synaptic vesicles is low (Parsons et al., 1993; Van der Kloot, 2003), a reduc- tion in VAChT abundance could result in electrophysio- logically ‘‘silent’’ vesicles, and thus a decrease in MEPP frequency. Prior experiments have demonstrated that overexpression of VAChT in immature Xenopus spinal neurons increases not only the amplitude but also the frequency of miniature excitatory postsynaptic currents (Song et al., 1997), indicating that, at least under certain conditions, VAChT expression levels can affect electro- physiological detection of exocytosis. Similarly, in Dro- sophila mutants with decreased neuromuscular expres- sion of the vesicular glutamate transporter, there are major deficits in frequency of miniature end-plate cur- rents, but no alterations in quantal size (Daniels et al., 2006). Remarkably, VAChT phosphorylation by PKC af- fects its trafficking to secretory vesicles, suggesting that alterations in VAChT expression in synaptic vesicles could occur physiologically (Krantz et al., 2000).

Transcription factors are key molecules that finely tune gene expression in response to injury. We focused on the role of a transcription factor, Foxn1, whose expression is limited to the skin and thymus epithelium. Our previous studies showed that Foxn1 inactivity in nude mice creates a pro-regenerative environment during skin wound healing. To explore the mechanistic role of Foxn1 in the skin wound healing process, we analyzed post-injured skin tissues from Foxn1::Egfp transgenic and C57BL/6 mice with Western Blotting, qRT- PCR, immunofluorescence and flow cytometric assays. Foxn1 expression in non-injured skin localized to the epidermis and hair follicles. Post-injured skin tissues showed an intense Foxn1-eGFP signal at the wound margin and in leading epithelial tongue, where it co-localized with keratin 16, a marker of activated keratinocytes. This data support the con- cept that suprabasal keratinocytes, expressing Foxn1, are key cells in the process of re-epi- thelialization. The occurrence of an epithelial-mesenchymal transition (EMT) was confirmed by high levels of Snail1 and Mmp-9 expression as well as through co-localization of vimen- tin/E-cadherin-positive cells in dermis tissue at four days post-wounding. Involvement of Foxn1 in the EMT process was verified by co-localization of Foxn1-eGFP cells with Snail1 in histological sections. Flow cytometric analysis showed the increase of double positive E- cadherin/N-cadherin cells within Foxn1-eGFP population of post-wounded skin cells iso- lates, which corroborated histological and gene expression analyses. Together, our findings indicate that Foxn1 acts as regulator of the skin wound healing process through engage- ment in re-epithelization and possible involvement in scar formation due to Foxn1 activity during the EMT process.

The calpain family of cysteine proteases have been found to have an important role in a diverse number of cellular events ranging from regulation of the actin cytoskeleton (reviewed in [6]) to modulation of apoptosis [7]. There are two ubiquitously expressed isoforms of calpain, calpain 1 (u) and calpain 2 (m), that are distinguished by their calcium requirements for activity. These isoforms form heterodimers with the small calpain subunit, calpain 4, which stabilizes calpain 1 and 2. In the absence of calpain 4, there is a loss of both calpain 1 and 2 expression and activity [8]. Knockout of calpain 4 is embryonic lethal [8], and fibroblasts obtained from calpain 4 knockout mice have been shown to have impaired migration [9] and altered responses to apoptotic stimuli [7].

demonstrated that both RANKL (mostly by ca - dherin 11-expressing synovial fibroblasts and CD3 T cells) and OPG were expressed in the inflamed synovium; the presence of osteoclasts precursors in the inflamed synovial tissue and that the factors needed to local osteoclastogenesis are present in the SpA synovium. There were no qualitative or quantitative differences in the expression of RAN- KL, OPG, and RANK between nonpsoriatic SpA, psoriatic SpA and RA synovium with the same de- gree of inflammation. They conclude that the rela - tive protection against bone erosion in SpA cannot be explained by differences of RANK/RANKL/OPG synovial expression, and that these factors expres- sion is disconnected from systemic and local in- flammation 22 .

Abstract: Public policies frequently are implemented simultaneously rather than in isolation. We estimate the impacts —and possible synergies—of a rural development project (Pro-Gavião) and the Brazilian conditional cash transfer program (Bolsa Família). In partnership with the State Government of Bahia, Pro-Gavião was an IFAD-supported rural development project in 13 contiguous municipalities between 1997 and 2005. Census tract level data were extracted for the analysis from the 1995-96 and 2006 Agricultural Censuses. The evaluation uses propensity score matching to construct a control group of untreated census tracts, and a difference-in-differences estimation to identify impacts. The outcomes analyzed include land productivity, agricultural income and child labor. Although Pro-Gavião involved significant investments in the region, the results suggest little if any program impact, or synergies between the two programs. The unexpected null findings are robust to alternative approaches to identifying the treated census tracts, matching techniques, and heterogeneity of impacts by initial level of poverty. We show that the lack of impacts is not driven by adverse rainfall in the treated communities, or the influence of other programs in the control communities. Alternative explanations for the null results are explored.

for accommodation of bas-relief panels [34]. So, there were no regularities to install the sculptures and "the whole sculpture was seen as one of the kinds of inner decor of the wall" [35, 81p]. According to G.A. Pugachenkova, the tradition of placing the royal statues in separately designated locations began from the era of Kanishka (in Surkh-Kotal the statues were located in inter- columnar spans) and presented a stylistic feature of the monumental art of late antiquity. In the "Hall of Kings" in Toprak-Kala, according to S.P. Tolstov, was a portrait gallery of Khoresm Siyavushids, "huge seated statues depicted the kings and the surrounding statues –their family members, and god-protectors" [36, 109p]. The isolated location of the figures symbolized the coming disintegration of the dynasty. However, it should be noted that "round sculpture, especially of large forms had no such strong roots in Central Asia" [3, 231p], as monumental painting, common even before the Hellenistic conquest. Wall and high relief compositions had dominated, particularly in the design of Buddhist monuments (stupas).

Human Papillomavirus cause a number of diseases most notably cervical cancer. K14- HPV16 transgenic mice expressing the HPV16 early genes in squamous epithelial cells provide a suitable experimental model for studying these diseases. MicroRNAs are small non-coding RNAs that play an important role in regulating gene expression and have been suggested to play an important role in cancer development. The role of miR-155 in cancer remains controversial and there is limited evidence linking this miRNA to HPV- associated diseases. We hypothesized that miR-155 expression modulates each tissue ’s susceptibility to develop HPV-associated carcinogenesis. In this study, we analyzed miR-155 expression in ear and chest skin samples from 22-26 weeks old, female K14-HPV16 transgenic (HPV16+/-) and wild-type (HPV-/-) mice. Among wild-type mice the expression of miR-155 was lower in ear skin compared with chest skin (p = 0.028). In transgenic animals, in situ carcinoma was present in all ear samples whereas chest tissues only showed epidermal hy- perplasia. Furthermore, in hyperplastic chest skin samples, miR-155 expression was lower than in normal chest skin (p = 0,026). These results suggest that miR-155 expression may modulate the microenvironmental susceptibility to cancer development and that high miR155 levels may be protective against the carcinogenesis induced by HPV16.

RANK belongs to the TNFR superfamily, is synthe- sized as a type I homotrimeric transmembrane protein, and is expressed by different tissues such as skeletal muscle, thymus, liver, colon, mamma- ry glands, prostate, pancreas, and cells of the monocyte/macrophage lineage (precursors and mature osteoclasts, B and T cells, dendritic cells, fi- broblasts, and articular chondrocytes). RANKL produced by osteoblasts binds to RANK in the sur- face of osteoclasts, recruits the tumor necrosis fac- tor receptor associated factor (TRAF) 2,5 and 6 that bind to RANK cytoplasmic domain (only TRAF6 seems to be essential in osteoclasts), leading to NF-kB activation and translocation to the nucleus. NF-kB increases c-Fos expression and c-Fos inte - racts with NFATc1 to trigger the osteoclastogenic genes transcription (Figure 2). At least seven signa - ling pathways are activated by RANK-mediated protein kinase signaling: four mediate osteoclas- togenesis (inhibitor of NF-kB kinase/NF-kB, c-Jun amino-terminal kinase/activator protein-1, c-myc, and calcineurin/NFATc1) and three mediate osteo - clast activation [Rous sarcoma oncogene (src) and mi togen-activated protein kinase kinase 6 (MKK6)/p38/microphthalmia-associated trans - cription factor (MITF)] and survival (src and ex- tracellular signal-regulated kinase) 1,2,6,8 .

plant species. Various pollination-control mechanisms are based on the genetic engineering of nuclear male sterility and its restoration had been reported and had emerged as tangible options for development of male sterile/restorer lines (Mariani et al., 1990; Rosellini et al., 2001; Jagannath et al., 2001, 2002; Bayer and Hess, 2005). Several strate- gies have been reported as producing NMS-plants in term of blocking pollen development by tissue-specific trans- gene expression(Mariani et al., 1990; Cho et al., 2001; Bur- gess et al., 2002),or altering specific metabolite levels in pollen development, such as sugars (Goetz et al., 2001), jasmonic acid (McConn and Browse, 1996; Stintzi and Browse, 2000) and flavonols (Fischer et al., 1997; Mayer et al., 2001).MS line sterility can be attributed to a newly in- troduced gene, Barnase, which encodes a special enzyme capable of cleaving RNA molecules in the cells, thereby leading to cell death. This ribonuclease is derived from Ba- cillus amyloliquefaciens (Hartley, 1988). To ensure that only male-flower parts are affected, Barnase should be linked to a special promoter activating the gene only in spe- cific cells responsible for the development of the male flower. As a result, either no pollen or no viable pollen is produced. This strategy had been successfully applied in obtaining male-sterile plants (Denis et al., 1993; Burgess et al., 2002; Luo et al., 2005). To date, most researchers have concentrated on creating RF lines containing a gene for an active substance (Barstar), which neutralizes Barnase