scholarly journals Mapping Transgene Insertion Sites Reveals Complex Interactions Between Mouse Transgenes and Neighboring Endogenous Genes

2018 ◽  
Author(s):  
Mallory A. Laboulaye ◽  
Xin Duan ◽  
Mu Qiao ◽  
Irene E. Whitney ◽  
Joshua R. Sanes
Keyword(s):  
Integration Site ◽  
Insertion Site ◽  
Cell Types ◽  
Regulatory Elements ◽  
Endogenous Gene ◽  
Complex Interactions ◽  
Distinct Cell ◽  
Transgenic Lines ◽  
Endogenous Genes ◽  
Insertion Sites

ABSTRACTTransgenic mouse lines are routinely employed to label and manipulate distinct cell types. The transgene generally comprises cell-type specific regulatory elements linked to a cDNA encoding a reporter or other proteins. However, off-target expression seemingly unrelated to the regulatory elements in the transgene is often observed, and sometimes suspected to reflect influences related to the site of transgene integration in the genome. To test this hypothesis, we used a proximity ligation-based method, Targeted Locus Amplification (TLA), to map the insertion sites of three well-characterized transgenes that appeared to exhibit insertion site-dependent expression in retina. The nearest endogenous genes to transgenes HB9-GFP, Mito-P, and TYW3 are Cdh6, Fat4 and Khdrbs2, respectively. For two lines, we demonstrate that expression reflects that of the closest endogenous gene (Fat4 and Cdh6), even though the distance between transgene and endogenous gene is 550 and 680 kb, respectively. In all three lines, the transgenes decrease expression of the neighboring endogenous genes. In each case, the affected endogenous gene was expressed in at least some of the cell types that the transgenic line has been used to mark and study. These results provide insights into the effects of transgenes and endogenous genes on each other’s expression, demonstrate that mapping insertion site is valuable for interpreting results obtained with transgenic lines, and indicate that TLA is a reliable method for integration site discovery.

scholarly journals NF-κB in Cancer Immunity: Friend or Foe?

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 355
Author(s):  
Guilhem Lalle ◽  
Julie Twardowski ◽  
Yenkel Grinberg-Bleyer
Keyword(s):  
Immune Responses ◽  
Therapeutic Potential ◽  
Cell Types ◽  
Transcriptional Activators ◽  
New Class ◽  
Complex Interactions ◽  
Cancer Immunity ◽  
Distinct Cell ◽  
Cancer Initiation ◽  
Cancer Outcome

The emergence of immunotherapies has definitely proven the tight relationship between malignant and immune cells, its impact on cancer outcome and its therapeutic potential. In this context, it is undoubtedly critical to decipher the transcriptional regulation of these complex interactions. Following early observations demonstrating the roles of NF-κB in cancer initiation and progression, a series of studies converge to establish NF-κB as a master regulator of immune responses to cancer. Importantly, NF-κB is a family of transcriptional activators and repressors that can act at different stages of cancer immunity. In this review, we provide an overview of the selective cell-intrinsic contributions of NF-κB to the distinct cell types that compose the tumor immune environment. We also propose a new view of NF-κB targeting drugs as a new class of immunotherapies for cancer.

scholarly journals Transcriptional Analysis of the Adeno-Associated Virus Integration Site

2009 ◽  
Vol 83 (23) ◽  
pp. 12512-12525 ◽  
Author(s):  
Nathalie Dutheil ◽  
Els Henckaerts ◽  
Erik Kohlbrenner ◽  
R. Michael Linden
Keyword(s):  
Transcriptional Activity ◽  
Integration Site ◽  
Regulatory Elements ◽  
Transcriptional Analysis ◽  
Start Codon ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Complex Interactions ◽  
Site Specific Integration ◽  
Virus Integration

ABSTRACT The nonpathogenic human adeno-associated virus type 2 (AAV-2) has adopted a unique mechanism to site-specifically integrate its genome into the human MBS85 gene, which is embedded in AAVS1 on chromosome 19. The fact that AAV has evolved to integrate into this ubiquitously transcribed region and that the chromosomal motifs required for integration are located a few nucleotides upstream of the translation initiation start codon of MBS85 suggests that the transcriptional activity of MBS85 might influence site-specific integration and thus might be involved in the evolution of this mechanism. In order to begin addressing this question, we initiated the characterization of the human MBS85 promoter region and compared its transcriptional activity to that of the AAV-2 p5 promoter. Our results clearly indicate that AAVS1 is defined by a complex transcriptional environment and that the MBS85 promoter shares key regulatory elements with the viral p5 promoter. Furthermore, we provide evidence for bidirectional MBS85 promoter activity and demonstrate that the minimal motifs required for AAV site-specific integration are present in the 5′ untranslated region of the gene and play a posttranscriptional role in the regulation of MBS85 expression. These findings should provide a framework to further elucidate the complex interactions between the virus and its cellular host in this unique pathway to latency.

scholarly journals Simultaneous profiling of multiple chromatin proteins in the same cells

2021 ◽  
Author(s):  
Sneha Gopalan ◽  
Yuqing Wang ◽  
Nicholas W. Harper ◽  
Manuel Garber ◽  
Thomas G Fazzio
Keyword(s):  
Rna Polymerase Ii ◽  
Direct Analysis ◽  
Cell Types ◽  
Regulatory Elements ◽  
Genome Wide ◽  
Distinct Cell ◽  
Direct Measurements ◽  
Cell Type Specific ◽  
Chromatin Proteins

Methods derived from CUT&RUN and CUT&Tag enable genome-wide mapping of the localization of proteins on chromatin from as few as one cell. These and other mapping approaches focus on one protein at a time, preventing direct measurements of co-localization of different chromatin proteins in the same cells and requiring prioritization of targets where samples are limiting. Here we describe multi-CUT&Tag, an adaptation of CUT&Tag that overcomes these hurdles by using antibody-specific barcodes to simultaneously map multiple proteins in the same cells. Highly specific multi-CUT&Tag maps of histone marks and RNA Polymerase II uncovered sites of co-localization in the same cells, active and repressed genes, and candidate cis-regulatory elements. Single-cell multi-CUT&Tag profiling facilitated identification of distinct cell types from a mixed population and characterization of cell type-specific chromatin architecture. In sum, multi-CUT&Tag increases the information content per cell of epigenomic maps, facilitating direct analysis of the interplay of different proteins on chromatin.

Automated Retroviral Insertion Site Sequence Analysis and Mapping Tool Followed by Database Analysis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5528-5528
Author(s):  
Stephanie Laufs ◽  
Frank A. Giordano ◽  
Agnes Hotz-Wagenblatt ◽  
Uwe Appelt ◽  
Daniel Lauterborn ◽  
...  
Keyword(s):  
Insertional Mutagenesis ◽  
Integration Site ◽  
Query Sequence ◽  
Cpg Islands ◽  
Insertion Site ◽  
High Throughput Analysis ◽  
Site Analysis ◽  
Conventional Analysis ◽  
Integration Site Analysis ◽  
Insertion Sites

Abstract Increasing use of retroviral vector-mediated gene transfer and recent reports on insertional mutagenesis in mice and humans created intense interest to characterize vector integrations on the genomic level. Techniques to determine insertion sites, mainly based on time consuming manual data processing and compilation, are thus commonly applied in gene therapy laboratories. Since a high variability in processing methods hampers further data comparison, there is an urgent need to systematically process the data arising from such analysis. The obtained sequences from the integration site analysis are judged to be authentic only if the matching part of the genomic query sequence is surrounded by the 5′LTR-sequence on the one side and the adapter-sequence on the other side. Therefore we developed an Integrationseq tool. In this task, different methods for converting the ABI sequence trace files to high quality sequences and for recognizing and deleting the LTR and adaptor parts of the isolated clones were implemented. If neither a primer nor a LTR could be found, the sequence is discarded. If the LTR is found on the complementary strand, the integration sequence is reversed. The remaining sequence between primer and LTR positions are taken as the n integration sequence and written to a sequence output file. We validated the Integrationseq tool using 259 trace files originating from integration site analysis (LM-PCR). Sequences can be trimmed by IntegrationSeq, leading to an increased yield of valid integration sequence detection, which has shown to be more sensitive (100%) than conventional analysis (94.3%) and 15 times faster than conventional analysis, while the specifities are equal (both 100%). Valid integration sequences get further processed with IntegrationMap for automatic genomic mapping. IntegrationMap runs 50 times faster than conventional methods and retrieves detailed information about whether integrations are located in or close to genes, the name of the gene, the exact localization in the transcriptional units and further parameters like the distance from the transcription start site to the integration. Further information, e.g. data about CpG-Islands, LINEs or SINEs, and their distances to the integration is also displayed. Output files generated by the task were found to be 99.8% identical with results retrieved by conventional mapping with the Ensembl alignment tool. Using both tools, IntegrationSeq and IntegrationMap, a validated, fast and standardized high-throughput analysis of insertion sites can be achieved for the first time.

scholarly journals Transmission pattern of hobo transposable element in transgenic lines of Drosophila melanogaster

1998 ◽  
Vol 71 (2) ◽  
pp. 97-107 ◽  
Author(s):  
VERONIQUE LADEVEZE ◽  
IBO GALINDO ◽  
NICOLE CHAMINADE ◽  
LUIS PASCUAL ◽  
GEORGES PERIQUET ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Population Studies ◽  
Insertion Site ◽  
Molecular Analyses ◽  
Transgenic Lines ◽  
Genomic Dnas ◽  
Insertion Sites ◽  
Method Show

This study is an attempt to trace the fate of hobo elements in the genomes of E strains of Drosophila melanogaster that have been transfected with pHFL1, a plasmid containing an autonomous hobo. Such long-term population studies (over 105 generations) could be very useful for better understanding the population and genomic dynamics of transposable elements and their pattern of insertions. Molecular analyses of hobo elements in the transfected lines were performed using Southern blots of XhoI-digested genomic DNAs. The complete element was observed in all six injected lines. In two lines we observed, at generation 100, two deleted elements, which did not correspond to Th1 and Th2. The results obtained by the in situ method show that the number of hybridization sites increases in each line and prove that the hobo element may be amplified in an RM genome. The hobo activity does not seem to be systematically correlated with the number of hobo elements. After generation 85, the evolution of the hobo element's insertion site number depends on the injected line. In all lines, the total number of insertions remains quite small, between 0 and 11. Hobo elements are located on each of the chromosomal arms. We describe ‘hotspots’ – insertion sites present in all lines and in all generations. On the 3R arm, a short inversion appeared once at generation 85.

scholarly journals Endocannabinoid Modulation of Nucleus Accumbens Microcircuitry and Terminal Dopamine Release

2021 ◽  
Vol 13 ◽  
Author(s):  
Dan P. Covey ◽  
Alyssa G. Yocky
Keyword(s):  
Nucleus Accumbens ◽  
Activity Patterns ◽  
Cell Types ◽  
Neural Architecture ◽  
Afferent Projections ◽  
Complex Interactions ◽  
Distinct Cell ◽  
Afferent Inputs ◽  
Control Terminal ◽  
Da Release

The nucleus accumbens (NAc) is located in the ventromedial portion of the striatum and is vital to valence-based predictions and motivated action. The neural architecture of the NAc allows for complex interactions between various cell types that filter incoming and outgoing information. Dopamine (DA) input serves a crucial role in modulating NAc function, but the mechanisms that control terminal DA release and its effect on NAc neurons continues to be elucidated. The endocannabinoid (eCB) system has emerged as an important filter of neural circuitry within the NAc that locally shapes terminal DA release through various cell type- and site-specific actions. Here, we will discuss how eCB signaling modulates terminal DA release by shaping the activity patterns of NAc neurons and their afferent inputs. We then discuss recent technological advancements that are capable of dissecting how distinct cell types, their afferent projections, and local neuromodulators influence valence-based actions.

scholarly journals Genetic reagents for making split-GAL4 lines in Drosophila

2017 ◽  
Author(s):  
Heather Dionne ◽  
Karen L. Hibbard ◽  
Amanda Cavallaro ◽  
Jui-Chun Kao ◽  
Gerald M. Rubin
Keyword(s):  
Nervous System ◽  
Genomic Dna ◽  
Expression Patterns ◽  
Cell Types ◽  
Biological Processes ◽  
Single Segment ◽  
Distinct Cell ◽  
Experimental Tool ◽  
Transgenic Lines

AbstractThe ability to reproducibly target expression of transgenes to small, defined subsets of cells is a key experimental tool for understanding many biological processes. The Drosophila nervous system contains thousands of distinct cell types and it has generally not been possible to limit expression to one or a few cell types when using a single segment of genomic DNA as an enhancer to drive expression. Intersectional methods, in which expression of the transgene only occurs where two different enhancers overlap in their expression patterns, can be used to achieve the desired specificity. This report describes a set of over 2,800 transgenic lines for use with the split-GAL4 intersectional method.

scholarly journals White as a reporter gene to detect transcriptional silencers specifying position-specific gene expression during Drosophila melanogaster eye development.

Genetics ◽  
1995 ◽  
Vol 141 (3) ◽  
pp. 1075-1086 ◽  
Author(s):  
Y H Sun ◽  
C J Tsai ◽  
M M Green ◽  
J L Chao ◽  
C T Yu ◽  
...  
Keyword(s):  
Pattern Formation ◽  
Expression Patterns ◽  
Insertion Site ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Drosophila Genome ◽  
Specific Expression ◽  
Endogenous Gene ◽  
Cis Acting ◽  
Cell Position

Abstract The white+ gene was used as a reporter to detect transcriptional silencer activity in the Drosophila genome. Changes in the spatial expression pattern of white were scored in the adult eye as nonuniform patterns of pigmentation. Thirty-six independent P[lacW] transposant lines were collected. These represent 12 distinct pigmentation patterns and probably 21 loci. The spatial pigmentation pattern is due to cis-acting suppression of white+ expression, and the suppression probably depends on cell position rather than cell type. The mechanism of suppression differs from inactivation by heterochromatin. In addition, activation of lacZ in P[lacW] occurs also in specific patterns in imaginal discs and embryos in many of the lines. The expression patterns of white+ and lacZ may reflect the activity of regulatory elements belonging to an endogenous gene near each P[lacW] insertion site. We speculate that these putative POSE (position-specific expression) genes may have a role in pattern formation of the eye as well as other imaginal structures. Three of the loci identified are optomotor-blind, engrailed and invected. teashirt is also implicated as a candidate gene. We propose that this "silencer trap"' may be an efficient way of identifying genes involved in imaginal pattern formation.

scholarly journals Mapping Transgene Insertion Sites Reveals the α-Cre Transgene Expression in Both Developing Retina and Olfactory Neurons

2021 ◽  
Author(s):  
Yimeng Fan ◽  
Wenyue Chen ◽  
Ran Wei ◽  
Wei Qiang ◽  
Joel Pearson ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Food Pellet ◽  
Insertion Site ◽  
Regulatory Elements ◽  
Rt Pcr ◽  
Olfactory Neurons ◽  
Reporter Mice ◽  
Insertion Sites ◽  
Olfactory Receptor Genes ◽  
Transgene Insertion

Abstract The Tg(Pax6-cre,GFP)2Pgr (α-Cre) mouse (MGI:3052661) is a commonly used Cre line thought to be retinal-specific. Using targeted locus amplification (TLA), we mapped the insertion site of the transgene, and defined primers useful to deduce zygosity. Further analyses revealed four tandem copies of the transgene. The insertion site mapped to clusters of vomeronasal and olfactory receptor genes. Using R26R and Ai14 Cre reporter mice, we confirmed retinal Cre activity, but also detected expression in olfactory neurons, implicating the influence of neighbouring regulatory elements. RT-PCR and the buried food pellet (BFP) test did not detect any effects of the transgene on flanking genes in the nasal mucosa and retina. Together, these data precisely map α-Cre, show that it does not affect surrounding loci, but reveal previously unanticipated transgene expression in olfactory neurons. The α-Cre mouse can be a valuable tool in both retinal and olfactory research.

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