scholarly journals Vector modifications to eliminate transposase expression following piggyBac-mediated transgenesis

2014 ◽  
Vol 4 (1) ◽  
Author(s):  
Syandan Chakraborty ◽  
HaYeun Ji ◽  
Jack Chen ◽  
Charles A. Gersbach ◽  
Kam W. Leong
Keyword(s):  
Transposase Expression

scholarly journals A hobo Transgene That Encodes the P-Element Transposase in Drosophila melanogaster: Autoregulation and Cytotype Control of Transposase Activity

Genetics ◽  
2002 ◽  
Vol 161 (1) ◽  
pp. 195-204 ◽  
Author(s):  
Michael J Simmons ◽  
Kevin J Haley ◽  
Craig D Grimes ◽  
John D Raymond ◽  
Jarad B Niemi
Keyword(s):  
Drosophila Melanogaster ◽  
Gonadal Dysgenesis ◽  
P Element ◽  
Hsp70 Gene ◽  
Alternate Splicing ◽  
Male And Female ◽  
P Elements ◽  
Male Germline ◽  
Female Germline ◽  
Transposase Expression

Abstract Drosophila were genetically transformed with a hobo transgene that contains a terminally truncated but otherwise complete P element fused to the promoter from the Drosophila hsp70 gene. Insertions of this H(hsp/CP) transgene on either of the major autosomes produced the P transposase in both the male and female germlines, but not in the soma. Heat-shock treatments significantly increased transposase activity in the female germline; in the male germline, these treatments had little effect. The transposase activity of two insertions of the H(hsp/CP) transgene was not significantly greater than their separate activities, and one insertion of this transgene reduced the transposase activity of P(ry+, Δ2-3)99B, a stable P transgene, in the germline as well as in the soma. These observations suggest that, through alternate splicing, the H(hsp/CP) transgene produces a repressor that feeds back negatively to regulate transposase expression or function in both the somatic and germline tissues. The H(hsp/CP) transgenes are able to induce gonadal dysgenesis when the transposase they encode has P-element targets to attack. However, this ability and the ability to induce P-element excisions are repressed by the P cytotype, a chromosomal/cytoplasmic state that regulates P elements in the germline.

scholarly journals Metaproteomics Reveals Abundant Transposase Expression in Mutualistic Endosymbionts

mBio ◽  
2013 ◽  
Vol 4 (3) ◽  
Author(s):  
Manuel Kleiner ◽  
Jacque C. Young ◽  
Manesh Shah ◽  
Nathan C. VerBerkmoes ◽  
Nicole Dubilier
Keyword(s):  
Transposable Elements ◽  
Recent Work ◽  
Current Model ◽  
Evolutionary Mechanisms ◽  
Host Environment ◽  
Underlying Causes ◽  
Mutagenic Effects ◽  
Current Paradigm ◽  
Rapid Emergence ◽  
Transposase Expression

ABSTRACTTransposases, enzymes that catalyze the movement of mobile genetic elements, are the most abundant genes in nature. While many bacteria encode an abundance of transposases in their genomes, the current paradigm is that the expression of transposase genes is tightly regulated and generally low due to its severe mutagenic effects. In the current study, we detected the highest number of transposase proteins ever reported in bacteria, in symbionts of the gutless marine wormOlavius algarvensiswith metaproteomics. At least 26 different transposases from 12 different families were detected, and genomic and proteomic analyses suggest that many of these are active. This high expression of transposases indicates that the mechanisms for their tight regulation have been disabled or no longer exist.IMPORTANCEThe expansion of transposable elements (TE) within the genomes of host-restricted symbionts and pathogens plays an important role in their emergence and evolution and might be a key mechanism for adaptation to the host environment. However, little is known so far about the underlying causes and evolutionary mechanisms of this TE expansion. The current model of genome evolution in host-restricted bacteria explains TE expansion within the confines of the paradigm that transposase expression is always low. However, recent work failed to verify this model. Based on our data, we hypothesize that increased transposase expression, which has not previously been described, may play a role in TE expansion, and could be one explanation for the sometimes very rapid emergence and evolution of new obligate symbionts and pathogens from facultative ones.

scholarly journals The autoregulation of a eukaryotic DNA transposon

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Corentin Claeys Bouuaert ◽  
Karen Lipkow ◽  
Steven S Andrews ◽  
Danxu Liu ◽  
Ronald Chalmers
Keyword(s):  
Binding Sites ◽  
Cell Biology ◽  
Eukaryotic Cell ◽  
Emergent Property ◽  
In Vivo Experiments ◽  
Constant Rate ◽  
Eukaryotic Dna ◽  
Transposase Expression

How do DNA transposons live in harmony with their hosts? Bacteria provide the only documented mechanisms for autoregulation, but these are incompatible with eukaryotic cell biology. Here we show that autoregulation of Hsmar1 operates during assembly of the transpososome and arises from the multimeric state of the transposase, mediated by a competition for binding sites. We explore the dynamics of a genomic invasion using a computer model, supported by in vitro and in vivo experiments, and show that amplification accelerates at first but then achieves a constant rate. The rate is proportional to the genome size and inversely proportional to transposase expression and its affinity for the transposon ends. Mariner transposons may therefore resist post-transcriptional silencing. Because regulation is an emergent property of the reaction it is resistant to selfish exploitation. The behavior of distantly related eukaryotic transposons is consistent with the same mechanism, which may therefore be widely applicable.

scholarly journals Somatic inactivation and reactivation of Ac associated with changes in cytosine methylation and transposase expression.

Genetics ◽  
1994 ◽  
Vol 138 (1) ◽  
pp. 213-225 ◽  
Author(s):  
T P Brutnell ◽  
S L Dellaporta
Keyword(s):  
Polymerase Chain Reaction ◽  
Cytosine Methylation ◽  
Methylation Profile ◽  
Open Reading Frame ◽  
Chain Reaction ◽  
Competitive Polymerase Chain Reaction ◽  
Reading Frame ◽  
Steady State Levels ◽  
Polymerase Chain ◽  
Transposase Expression

Abstract Several metastable Ac alleles at the maize p locus were identified that produced novel pericarp variegation patterns. From the transmission analysis of pericarp sectors, we show that Ac inactivation is a somatic process. Ac excision from P and transactivation of an unlinked Ds was delayed or absent in plants with metastable Ac alleles. These decreases in Ac activity were correlated to increases in cytosine methylation at specific sites near the start of Ac transcription (open reading frame a; ORFa) and at sites in some flanking P sequence. Reactivation of inactive alleles was accompanied by decreased methylation of Ac and P sequences. Using a competitive polymerase chain reaction assay, steady state levels of ORFa transcript were quantitatively compared among the various metastable alleles. We propose that changes in the methylation profile of Ac correspond to changes in Ac activity through the differential accumulation of Ac transcript.

scholarly journals A series of constitutive expression vectors to accurately measure the rate of DNA transposition and correct for auto-inhibition

2018 ◽  
Author(s):  
Michael Tellier ◽  
Ronald Chalmers
Keyword(s):  
Dna Sequences ◽  
Constitutive Expression ◽  
Expression Vectors ◽  
Single Chain ◽  
Dimer Interface ◽  
Inducible Promoters ◽  
Wide Range ◽  
Genomic Damage ◽  
Screening Assays ◽  
Transposase Expression

AbstractBackgroundTransposable elements (TEs) form a diverse group of DNA sequences encoding functions for their own mobility. This ability has been exploited as a powerful tool for molecular biology and genomics techniques. However, their use is sometimes limited because their activity is auto-regulated to allow them to cohabit within their hosts without causing excessive genomic damage. To overcome these limitations, it is important to develop efficient and simple screening assays for hyperactive transposases.ResultsTo widen the range of transposase expression normally accessible with inducible promoters, we have constructed a set of vectors based on constitutive promoters of different strengths. We characterized and validated our expression vectors with Hsmar1, a member of the mariner transposon family. We observed the highest rate of transposition with the weakest promoters. We went on to investigate the effects of mutations in the Hsmar1 transposase dimer interface and of covalently linking two transposase monomers in a single-chain dimer. We also tested the severity of mutations in the lineage leading to the human SETMAR gene, in which one copy of the Hsmar1 transposase has contributed a domain.ConclusionsWe generated a set of vectors to provide a wide range of transposase expression which will be useful for screening libraries of transposase mutants. We also found that mutations in the Hsmar1 dimer interface provides resistance to overproduction inhibition in bacteria, which could be valuable for improving bacterial transposon mutagenesis techniques.

332 INDUCIBLE RED FLUORESCENT PROTEIN (RFP) EXPRESSION IN PORCINE FIBROBLASTS AND TRANSGENIC CLONED EMBRYOS USING piggyBac TRANSPOSITION

2011 ◽  
Vol 23 (1) ◽  
pp. 262 ◽  
Author(s):  
S. J. Kim ◽  
O. J. Koo ◽  
S. J. Park ◽  
J. H. Moon ◽  
D. K. Kwon ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Plasmid Vector ◽  
Transgenic Pigs ◽  
Donor Cells ◽  
Transgenic Cells ◽  
Fluorescence Protein ◽  
Red Fluorescence Protein ◽  
Animal Resources ◽  
Retrovirus Infection ◽  
Transposase Expression

Transgenic pigs are promising animal resources for human disease models and organ donors for xenotransplantation, because they resemble humans anatomically and physiologically. Transgenic pigs have been produced from transfected donor cells using several gene delivery systems including retrovirus infection. Recently, it has been reported that piggyBac (PB) transposition is a highly efficient tool in producing transgenic mice. This study investigated the use of PB transposition to establish transgenic cells and produce transgenic cloned embryos in pigs. We constructed plasmid DNA with red fluorescence protein (RFP) expressed by tetracycline-dependent cassette (from Addgene) with PB site using gateway cloning. We co-transfected porcine fibroblasts with the structured plasmid vector (pB-TET-DsRed), pB-rtTA (from Addgene), and a transposase expression vector pCy43 (Sanger Insitute, Hinxton, UK) using Fugene HD. After 24 h, 2 μg mL–1 doxycycline was added to the culture medium to turn on RFP expression. After 48 h of culture, 1 mg mL–1 neomycin was added to select stable RFP transfectants. Selected fibroblasts were cultured for 9 days without doxycycline, thus reducing RFP expression. After establishment of inducible RFP-expressing cells, the cells were used for somatic cell nuclear transfer. Embryos were cultured in porcine zygote medium-3, and 2 μg mL–1 doxycycline was added 5 days later. As a result, RFP expression was detected in the blastocysts. In conclusion, this study demonstrated that the inducible RFP gene in porcine fibroblasts and embryos was controlled by PB transposition system. Furthermore, this system could be a means of delivering an exogenous gene into porcine somatic cells and embryos for transgenic research. This study was supported by grants from MKE (#2009-67-10033839, #2009-67-10033805), IPET (#109023-05-1-CG000), NRF (#M10625030005-10N250300510), and BK21 program.

Regulation of IS10 Transposase Expression by RNA/RNA Pairing

1987 ◽  
pp. 413-421 ◽  
Author(s):  
N. KLECKNER ◽  
J.D. KITTLE ◽  
R.W. SIMONS
Keyword(s):  
Transposase Expression
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