Estudos realizados no Laboratório de Drosophila da UFRGS tem caracterizado linhagens de Drosophila willistoni quanto à presença de Elementos Transponíveis (TEs) e à existência do fenômeno de Disgenesia Híbrida nesta espécie. Como conseqüência destes estudos, o presente trabalho se propôs a ampliar o conhecimento sobre o papel destes elementos na geração de variabilidade nesta espécie e abordou os TEs P, hobo, gypsy e 412, anteriormente identificados no genoma de D. willistoni, quanto à sua atividade transcricional. Em nosso trabalho, verificamos a presença de transcritos de P, gypsy e 412 em adultos, sugerindo uma regulação pós-transcricional destes elementos, como já sugerido para o elemento P, considerando que as linhagens utilizadas não se caracterizam pela hipermutabilidade. Devido à descrição prévia da Síndrome da Disgenesia do Híbrido na prole do cruzamento entre as linhagens 17A2 e Wip de D. willistoni, foi estabelecido o padrão de expressão do elemento P durante o desenvolvimento embrionário das duas linhagens. O padrão de expressão embrionário em D. melanogaster também foi estabelecido, para fins comparativos, uma vez que se aceita a ocorrência de um evento de transferência horizontal de P de D. willistoni para D. melanogaster. A similaridade entre os padrões de expressão nas duas espécies, sugere que o elemento P é regulado pelo seu próprio promotor e que não é dependente de promotores de genes vizinhos aos seus sítios de inserção. Foi estabelecida a presença de transcritos potenciais da transposase e de um repressor da transposição de P nos embriões analisados. Além do transcrito correspondente ao repressor, que é gerado por processamento alternativo do transcrito da transposase, obteve-se indícios da presença de transcritos antisenso do próprio elemento nos embriões, sugerindo a regulação por interferência de RNA (RNAi) neste estágio do desenvolvimento de Drosophila. Diferenças transcricionais do elemento P entre D. willistoni e D. melanogaster, estão relacionadas ao número de transcritos deletados de P que são expressos em maior número em D. melanogaster do que em D. willistoni, corroborando a idéia da invasão recente do genoma da primeira por este elemento. A expressão dos TEs descrita neste trabalho relata a regulação complexa destes elementos, evidenciando a importância da continuidade deste estudo. / Studies accomplished in the Laboratory of Drosophila of UFRGS have been characterizing strains of Drosophila willistoni in respect to the presence of Transposable Elements (TEs) and to the occurrence of the Hybrid Dysgenesis phenomenon in this species. As a consequence of these studies, the present work aimed to broaden the knowledge about the role of these elements in the genesis of variability in this species, by approaching the transcriptional expression of P, hobo, gypsy and 412 TEs, already described in the D. willistoni genome. In our work, we verified the presence of P, gypsy, and 412 transcripts in adults, suggesting post-transcriptional regulation, like already described for P element in D. melanogaster, considering that the strains studied were not characterized by hypermutability. Due to the previous description of the Hybrid Dysgenesis Syndrome in the offspring resulting of crosses between 17A2 and Wip D. willistoni strains, their P element expression pattern during the embryonic development was established. The embryonic P element expression pattern in D. melanogaster was also established, for comparative purpose, since the occurrence of a horizontal transfer event of this element from D. willistoni to D. melanogaster is accepted. The similarity among these expression patterns in both species suggests that P element is regulated by its own promoter and that it’s not dependent of the insertion sites neighboring genes promoters. The presence of putative P element transcripts of transposase and of the transposase repressor was established in the analyzed embryos. Besides the transposase repressor transcript, that is result of an alternative splicing of the transposase transcript, it was obtained indication of the presence of antisense transcripts of P element in the embryos, suggesting the regulation by RNA interference (RNAi) in this stage of development of Drosophila. Transcriptional differences of the P element between D. willistoni and D. melanogaster, are related to the number of deleted transcripts of P that are expressed in larger number in D. melanogaster than in D. willistoni, corroborating the idea of the recent invasion of the genome of the first species by this element. The expression of TEs described in this work suggests a complex regulation of these elements, evidencing the importance of the continuity of this study.

Estudos realizados no Laboratório de Drosophila da UFRGS tem caracterizado linhagens de Drosophila willistoni quanto à presença de Elementos Transponíveis (TEs) e à existência do fenômeno de Disgenesia Híbrida nesta espécie. Como conseqüência destes estudos, o presente trabalho se propôs a ampliar o conhecimento sobre o papel destes elementos na geração de variabilidade nesta espécie e abordou os TEs P, hobo, gypsy e 412, anteriormente identificados no genoma de D. willistoni, quanto à sua atividade transcricional. Em nosso trabalho, verificamos a presença de transcritos de P, gypsy e 412 em adultos, sugerindo uma regulação pós-transcricional destes elementos, como já sugerido para o elemento P, considerando que as linhagens utilizadas não se caracterizam pela hipermutabilidade. Devido à descrição prévia da Síndrome da Disgenesia do Híbrido na prole do cruzamento entre as linhagens 17A2 e Wip de D. willistoni, foi estabelecido o padrão de expressão do elemento P durante o desenvolvimento embrionário das duas linhagens. O padrão de expressão embrionário em D. melanogaster também foi estabelecido, para fins comparativos, uma vez que se aceita a ocorrência de um evento de transferência horizontal de P de D. willistoni para D. melanogaster. A similaridade entre os padrões de expressão nas duas espécies, sugere que o elemento P é regulado pelo seu próprio promotor e que não é dependente de promotores de genes vizinhos aos seus sítios de inserção. Foi estabelecida a presença de transcritos potenciais da transposase e de um repressor da transposição de P nos embriões analisados. Além do transcrito correspondente ao repressor, que é gerado por processamento alternativo do transcrito da transposase, obteve-se indícios da presença de transcritos antisenso do próprio elemento nos embriões, sugerindo a regulação por interferência de RNA (RNAi) neste estágio do desenvolvimento de Drosophila. Diferenças transcricionais do elemento P entre D. willistoni e D. melanogaster, estão relacionadas ao número de transcritos deletados de P que são expressos em maior número em D. melanogaster do que em D. willistoni, corroborando a idéia da invasão recente do genoma da primeira por este elemento. A expressão dos TEs descrita neste trabalho relata a regulação complexa destes elementos, evidenciando a importância da continuidade deste estudo. / Studies accomplished in the Laboratory of Drosophila of UFRGS have been characterizing strains of Drosophila willistoni in respect to the presence of Transposable Elements (TEs) and to the occurrence of the Hybrid Dysgenesis phenomenon in this species. As a consequence of these studies, the present work aimed to broaden the knowledge about the role of these elements in the genesis of variability in this species, by approaching the transcriptional expression of P, hobo, gypsy and 412 TEs, already described in the D. willistoni genome. In our work, we verified the presence of P, gypsy, and 412 transcripts in adults, suggesting post-transcriptional regulation, like already described for P element in D. melanogaster, considering that the strains studied were not characterized by hypermutability. Due to the previous description of the Hybrid Dysgenesis Syndrome in the offspring resulting of crosses between 17A2 and Wip D. willistoni strains, their P element expression pattern during the embryonic development was established. The embryonic P element expression pattern in D. melanogaster was also established, for comparative purpose, since the occurrence of a horizontal transfer event of this element from D. willistoni to D. melanogaster is accepted. The similarity among these expression patterns in both species suggests that P element is regulated by its own promoter and that it’s not dependent of the insertion sites neighboring genes promoters. The presence of putative P element transcripts of transposase and of the transposase repressor was established in the analyzed embryos. Besides the transposase repressor transcript, that is result of an alternative splicing of the transposase transcript, it was obtained indication of the presence of antisense transcripts of P element in the embryos, suggesting the regulation by RNA interference (RNAi) in this stage of development of Drosophila. Transcriptional differences of the P element between D. willistoni and D. melanogaster, are related to the number of deleted transcripts of P that are expressed in larger number in D. melanogaster than in D. willistoni, corroborating the idea of the recent invasion of the genome of the first species by this element. The expression of TEs described in this work suggests a complex regulation of these elements, evidencing the importance of the continuity of this study.

Gene therapy is an attractive treatment for many genetic diseases because rather than treat the symptoms of the disease, it has the potential to correct the underlying defect. Cystic fibrosis and hemophilia A are two monogenic disorders that are particularly well-suited to treatment with gene therapy as a relatively small increase in the function is needed to see improvement. Gene therapy has provided some correction in both diseases using a variety of vector systems but sustained expression and long term correction have yet to be demonstrated in the clinic. It is unclear in which cell type(s) correction of the underlying defect in cystic fibrosis will be most effective. Studies indicate that the majority of CFTR expression is in the submucosal glands and ciliated epithelia – a terminally differentiated cell type (Engelhardt, J.F. et al, 2004, Journal of Clinical Investigation). Therapeutic gene transfer would thus be most effective if achieved in a progenitor cell type. Additionally, the native regulation of CFTR has not been definitively elucidated. To this end, one goal of our studies is to develop a lentiviral vector system with heterologous promoters of varying strengths and cell specificity to aid in our selection of optimal reagents for appropriate CFTR expression. We show that use of novel internal promoters from the human PLUNC and WDR65 genes direct persistent expression in the airway. Additionally, disruption of the nasal epithelium with the detergent polidocanol eliminated reporter expression in mouse airway. Two weeks post-treatment, expression returned indicating targeting of a progenitor cell population with our novel vectors. Integrating vector systems can treat chronic diseases such as cystic fibrosis because expression can persist long term from these vectors if cells with progenitor capacity are targeted (Sinn, P.L. et al, 2005, Journal of Virology). However, the potential for genotoxicity from vector-related dysregulation is a concern. Thus, a second aim of these studies was to develop a lentiviral vector that can target a specific locus in the genome. We developed a FIV vector in which the integrase was modified with a protein-binding domain that when co-delivered with a fusion consisting of the cognate protein and a DNA binding domain would tether the vector to the appropriate locus. Unfortunately, integrase modification rendered the vector catalytically inactive. Lastly, we hoped to develop a non-viral transposon vector system (piggyBac) for gene transfer applications to the liver for treatment of hemophilia A. The recent demonstration that piggyBac transposase is highly active in mammalian cells warrants further development of this vector as an alternative to other non-viral integrating vector systems currently under investigation. We showed persistent reporter and therapeutic transgene expression in the livers of mice treated with the piggyBac vector. Furthermore, we show for the first time in vivo persistence and increased expression from the recently developed hyperactive transposase. The development of integrating vectors targeted to specific tissues or genomic loci is important in for treatment of the monogenic diseases cystic fibrosis and hemophilia A.

Neisseria gonorrhoeae is a human-specific pathogen that causes gonorrhea. It is defined as a super bacterium by the WHO due to the emergence of gonococci that are resistant to a variety of antibiotics and a rapidly increasing infection incidence. Genome-wide investigation of neisserial gene essentiality and novel virulence factors is urgently required in order to identify new targets for anti-neisserial therapeutics. To identify essential genes and new virulence factors, a high-density mutant library in N. gonorrhoeae MS11 was generated by in vitro transposon mutagenesis. The transposon library harbors more than 100,000 individual mutants, a density that is unprecedented in gonococcal research. Essential genes in N. gonorrhoeae were determined by enumerating frequencies of transposon insertion sites (TIS) with Illumina deep sequencing (Tn-seq). Tn-seq indicated an average distance between adjacent TIS of 25 bp. Statistical analysis unequivocally demonstrated 781 genes that were significantly depleted in TIS and thus are essential for Neisseria survival. A subset of the genes was experimentally verified to comprise essential genes and thus support the outcome of the study. The hereby identified candidate essential genes thus may constitute excellent targets for the development of new antibiotics or vaccines. In a second study, the transposon mutant library was applied in a genome-scale “negative-selection strategy” to identify genes that are involved in low phosphate-dependent invasion (LPDI). LPDI is dependent on the Neisseria porin subtype PorBIA which acts as an epithelial cell invasin in absence of phosphate and is associated with severe pathogenicity in disseminated gonococcal infections (DGI). Tn-seq demonstrated 98 genes, which were involved in adherence to host cells and 43 genes involved in host cell invasion. E.g. the hypothetical protein NGFG_00506, an ABC transporter ATP-binding protein NGFG_01643, as well as NGFG_04218 encoding a homolog of mafI in N. gonorrhoeae FA1090 were experimentally verified as new invasive factors in LPDI. NGFG_01605, a predicted protease, was identified to be a common factor involved in PorBIA, Opa50 and Opa57-mediated neisserial engulfment by the epithelial cells. Thus, this first systematic Tn-seq application in N. gonorrhoeae identified a set of previously unknown N. gonorrhoeae invasive factors which demonstrate molecular mechanisms of DGI. / Neisseria gonorrhoeae ist ein human-spezifisches Pathogen, das die Krankheit Gonorrhoe verursacht. Aufgrund der steigenden Anzahl antibiotikaresistenter Gonokokken und der damit verbundenen, rapide zunehmenden Anzahl von Infektionen erklärte die WHO Gonokokken 2012 zum Superbakterium. Daher ist eine genomweite Untersuchung der neisseriellen Genessentiatialität und neuer Virulenzfaktoren dringend erforderlich, um neue Ziele für die antineisserielle Therapie zu identifizieren. Hierzu wurde eine high-density Mutantenbibliothek in N. gonorrhoeae MS11 durch in vitro Transposonmutagenese generiert. Die Transposonbibliothek enthält mehr als 100.000 individuelle Mutanten - eine Dichte, die in der Gonokokken-Forschung beispiellos ist. Essentielle Gene von N. gonorrhoeae wurden durch die Ermittlung der Häufigkeit von Transposon insertion sites (TIS) mit Hilfe von Illumina deep sequencing (Tn-seq) bestimmt. Tn-seq ergab eine durchschnittliche Distanz von 25 Basenpaaren zwischen benachbarten TIS. Die statistische Analyse zeigte eindeutig 781 Gene, die signifikant weniger TIS aufwiesen und deshalb als essentiell für das Überleben der Neisserien verstanden werden können. Für ausgewählte Gene wurde experimentell bestätigt, dass sie essentielle Gene beinhalten, wodurch das Ergebnis der Tn-seq unterstützt wird. Die hierbei identifizierten essentiellen Gene könnten exzellente Targets für die Entwicklung neuer Antibiotika oder Impfstoffe darstellen. In einer zweiten Studie wurde die Transposon Mutanten Bibliothek für eine genomweite „negative Selektionsstrategie“ bereitgestellt. Es sollten Gene identifiziert werden, die an der phosphatfreien Invasion (low phosphate-dependent invasion = LPDI) beteiligt sind. Die LPDI ist vom neisseriellen Porin Subtyp PorBIA abhängig, welches bei Epithelzellen in Abwesenheit von Phosphat als Invasin fungiert und mit einer schweren Pathogenität in disseminierenden Gonokokkeninfektionen (DGI) assoziiert ist. Tn-seq ergab 98 Gene, die an der Adhärenz an die Wirtszelle, und 43 Gene, die an der Wirtszellinvasion beteiligt waren. Zum Beispiel wurden das hypothetische Protein NGFG_00506, ein ABC Transporter, das ATP-bindende Protein NGFG_01643, wie auch NGFG_04218, das für ein Homolog von mafI in N. gonorrhoeae FA1090 kodiert, experimentell als neue Invasionsfaktoren in der LPDI verifiziert. NGFG_01605, bei dem angenommen wird, dass es sich um eine Protease handelt, wurde als ein allgemeiner Faktor identifiziert, der an der PorBIA-, Opa50- and Opa57-vermittelten Einstülpung der Membran von Epithelzellen beteiligt ist. Die erste systematische Anwendung von Tn-seq in N. gonorrhoeae identifizierte eine Reihe bisher unbekannter Invasionsfaktoren von N. gonorrhoeae, die molekulare Mechanismen der DGI zeigen.

We demonstrate that hydrogen production can be increased by random mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and that hydrogen production can be further increased in the chemically-mutagenized strain by targeted gene deletion and overexpression of genes related to formate metabolism. Chemical mutagenesis of Escherichia coli BW25113 hyaB hybC hycE::kan/pBS(Kan)-HycE to form strain 3/86 resulted in 109 +/- 0.5- fold more hydrogen; 3/86 lacks functional hydrogen uptake hydrogenases 1 and 2, has hydrogenproducing hydrogenase 3 inactivated from the chromosome, and has constitutively active hydrogenase 3 based on expression of the large subunit of hydrogenase 3 from a high copy number plasmid. Deleting fdoG, which encodes formate dehydrogenase O, (that diverts formate from hydrogen), from chemical mutagen 3/86 increased hydrogen production 188 +/- 0.50-fold (relative to the unmutagenized strain), and deletion of hycA, which encodes the repressor of formate hydrogen lyase (FHL), increased hydrogen production 232 +/- 0.50-fold. Deleting both fdoG and hycA increased hydrogen production 257 +/- 0.50-fold, and overexpressing fhlA along with the fdoG hycA mutations increased hydrogen 308 +/- 0.52-fold. Whole-transcriptome analysis of chemical mutagen 3/86 revealed 89 genes were induced and 31 genes were repressed. In an effort to identify chromosomal mutations in chemical mutagen 3/86, we performed comparative genome sequencing and identified two chromosomal loci with mutations in coding regions of ftnA and yebJ; however, neither gene was related to the increased hydrogen production as determined by the close vial (short) hydrogen assay. In addition, transposon mutagenesis, which is one of the most efficient strategies for creating random mutations in the genomic DNA, was performed in two different strains: E. coli BW25113 hyaB hybC hycA fdoG::kan/pCA24N-FhlA and E. coli MG1655 to identify beneficial mutations for hydrogen production. As a result of screening 461 E. coli BW25113 hyaB hybC hycA fdoG::kan/pCA24N-FhlA transformants and 1000 E. coli MG1655 transformants, three interesting mutations have been discovered in E. coli BW25113 hyaB hybC hycA fdoG::kan/pCA24N-FhlA transformants (gpsA, dipZ, glgP) and 1 beneficial mutation in E. coli MG1655 transformants (malT). When any of these genes gpsA, dipZ, or glgP is disrupted by Tn5 insertion, hydrogen production decreases 17, 3 and 8-fold, respectively. Additionally, when malT gene is disrupted by Tn5 insertion, hydrogen increases 3.4-fold.

Techn. Univ., Diss., 2009--München.

Universiẗat, Diss., 2003--Bielefeld.

The rise of antibiotic resistance in common human pathogens and the lack of development of novel therapeutic treatments has created a threat to global health. A unique source for potential novel treatments are from microorganisms, particularly within the complex, antagonistic polymicrobial interactions that take place in microbial communities. These unique mechanisms utilized by microorganisms to fight each other could potentially identify novel therapeutic targets for use at a clinical level, however, there is a lack of research in this area to determine its applicability. Alcaligenes faecalis is a Gram-negative bacterium that seldom causes human disease and has been observed in our lab to show competitive, contact-dependent inhibitory mechanisms against Bacillus species, Candida albicans, and Staphylococcus species. These bacterial and eukaryotic microbes are increasingly a common source of human disease and all exhibit increased incidences of drug resistance. In this study, genetic elements related to A. faecalis’ contact-dependent inhibitory mechanism were determined via transposon mutagenesis. Genomic sequencing was performed on mutant strains of A. faecalis that exhibited diminished inhibition or loss-of-function inhibition against the competing microbes. Four of these A. faecalis mutant strains were successfully sequenced and compared to NCBI’s genomic database. The proteins of the interrupted genetic elements were identified as a FAD-binding oxidoreductase, MFS transporter, and mechanosensitive ion channel. Further analysis of these mutants is needed to determine their role in the mechanism of A. faecalis’ antimicrobial activity. The findings of this study may aid in the identification of new therapeutic targets for novel S. aureus, C. albicans, and Bacillus species treatments.

The rise of antibiotic resistance of common human pathogens and the lack of development of novel therapeutic treatments has created a threat to global health. A unique source for potential novel treatments are from microorganisms, particularly within the complex, antagonistic polymicrobial interactions that take place in microbial communities. These unique mechanisms utilized by microorganisms to fight each other could potentially identify novel therapeutic targets for use at a clinical level, however, there is a lack of research in this area to determine its applicability. Alcaligenes faecalis is a Gram-negative bacterium that seldom causes human disease and has been observed in our lab to show competitive, contact-dependent inhibitory mechanisms against Bacillus species, Candida albicans, and Staphylococcus species. These bacterial and eukaryotic microbes are increasingly a common source of human disease and all exhibit increased incidences of drug resistance. In this study, genetic elements related to A. faecalis’ contact-dependent inhibitory mechanism were determined via transposon mutagenesis. Genomic sequencing was performed on mutant strains of A. faecalis that exhibited diminished inhibition or loss-of-function inhibition of the competing microbes. In A. faecalis mutant strains P2-9 and P1-42, the interrupted gene was identified as a FAD-binding oxidoreductase with a 94% and 90% match of nucleotide sequence. Mutant strain P2-25’s interrupted gene was identified as an MFS transporter with a 100% match and P2-30’s interrupted gene was identified as a mechanosensitive ion channel with a 100% match. Further analysis of these mutants is needed to determine their role in the mechanism of A. faecalis’ antimicrobial activity. The findings of this study may aid in the identification of new therapeutic targets for novel S. aureus, C. albicans, and Bacillus treatments.

Das Multiple Myelom (MM) ist ungeachtet neuer medikamentöser Therapien weiterhin eine für die allermeisten Patienten unheilbare Erkrankung. Aktuelle Whole-Genome-Sequenzierungen konnten die Annahme, dass hierfür die genetische Heterogenität des MM verantwortlich ist, bestätigen. Um dieses onkogene Signalnetzwerk auf effiziente Weise zu untersuchen, wurde ein induzierbares Knock-down-System basierend auf der weitverbreiteten RNA-Interferenz und dem neuen, innovativen Sleeping Beauty Transposon System (SBTS) entwickelt. Die Etablierung des SBTS im MM zeigte, dass die CMV-Promotor-vermittelte EGFP-Expression über 231 Tage stabil ist. Die Verwendung des SBTS ermöglicht es, Zellen bei niedrigster biologischer Schutzstufe (S1) stabil zu transfizieren. Am Beispiel des MAPK-Signalweges konnten stabile shRNA-vermittelte Knock-downs in verschiedenen MM-Zelllinien erfolgreich durchgeführt werden. Um ein induzierbares Tet-On-System zur shRNA-Expression zu erstellen, wurden zum einen dem verwendeten H1-Promotor zwei TetO-Sequenzen hinzugefügt. Zum anderen wurde durch die Verwendung eines zweiten, neu konstruierten SBTS Vektors mit anderer Selektionsresistenz in den verwendeten Zellen das Tet-Repressor-Protein (TetR) stabil exprimiert. Die notwendige Expression von TetR konnte nur mittels CAG-Promotors erreicht werden. Am Beispiel von ERK2 konnte gezeigt werden, dass es durch die stabile Transfektion und die Induktion des Knock-downs mit Doxycyclin möglich ist, den Knock-down Effekt zu erzielen. Das etablierte Plasmid-basierte System stellt somit ein versatiles, einfach anwendbares, kostengünstiges und zeitsparendes Werkzeug dar, induzierbare Knock-downs durch RNA-Interferenz für einzelne oder mehrere Proteine gleichzeitig zu erzielen. Es ist davon auszugehen, dass die Anwendung aufgrund der Verwendung etablierter Techniken und der leichten Modifizierbarkeit nicht nur auf das MM begrenzt sein wird. / Multiple Myeloma (MM) is regardless of new drug therapies still an incurable disease for the majority of patients. Large-scale whole-genome-sequencing studies strongly support the assumption that the reason for this is the large genetic heterogeneity of MM. To gain knowledge about the complex oncogenetic signalling efficiently, in this work, a system for inducible knock-downs based on the wide-spread RNA-interference and the new innovative Sleeping Beauty Transposon System (SBTS) was developed. The successful establishment of the SBTS in MM revealed that EGFP-expression by a CMV-promotor is stable for at least 231 days without any further selection by antibiotics. The use of SBTS makes it possible to transfect cells stably at the lowest biological safety level (S1). Utilizing the MAPK-signal path as an example it was possible to successfully demonstrate shRNA-provided knock-downs for up to four targets at once in several different MM-cell lines. To create an inducible Tet-on-system for shRNA-Expression, two TetO-sequences were added to the used H1-Promotor. Furthermore, the required Tet-repressor-protein (TetR) was expressed by another newly constructed SBTS vector, which contains another selection resistance. After trying different approaches, it was possible to express the necessary amount of TetR by using the CAG-Promotor, which was verified by Western Blot. Using ERK2 as a sample target, it was possible to show that with stable transfection and induction of the knock-down with doxycycline it is possible to measure knock-down results without the toxic side effects of the electroporation necessary for transfection. The established plasmid-based system serves as an easily applicable, cost-efficient and time-saving tool for inducible knock-downs by RNAi for single or multiple proteins. Due to the fact that yet established techniques were used and thanks to the simplicity of the customization it is assumed that the application is not only limited to MM.