scholarly journals Mobilization and Mechanism of Transcription of Integrated Self-Inactivating Lentiviral Vectors

2005 ◽  
Vol 79 (13) ◽  
pp. 8410-8421 ◽  
Author(s):  
Hideki Hanawa ◽  
Derek A. Persons ◽  
Arthur W. Nienhuis
Keyword(s):  
Lentiviral Vectors ◽  
Regulatory Elements ◽  
Trap Design ◽  
Promoter Sequences ◽  
Vector Integration ◽  
Vector Genome ◽  
Promoter Trap ◽  
A Cell ◽  
Or Gene ◽  
Cryptic Promoters

ABSTRACT Permanent genetic modification of replicating primitive hematopoietic cells by an integrated vector has many potential therapeutic applications. Both oncoretroviral and lentiviral vectors have a predilection for integration into transcriptionally active genes, creating the potential for promoter activation or gene disruption. The use of self-inactivating (SIN) vectors in which a deletion of the enhancer and promoter sequences from the 3′ long terminal repeat (LTR) is copied over into the 5′ LTR during vector integration is designed to improve safety by reducing the risk of mobilization of the vector genome and the influence of the LTR on nearby cellular promoters. Our results indicate that SIN vectors are mobilized in cells expressing lentiviral proteins, with the frequency of mobilization influenced by features of the vector design. The mechanism of transcription of integrated vector genomes was evaluated using a promoter trap design with a vector encoding tat but lacking an upstream promoter in a cell line in which drug resistance depended on tat expression. In six clones studied, all transcripts originated from cryptic promoters either upstream or within the vector genome. We estimate that approximately 1 in 3,000 integrated vector genomes is transcribed, leading to the inference that activation of cryptic promoters must depend on local features of chromatin structure and the constellation of nearby regulatory elements as well as the nature of the regulatory elements within the vector.

Diminished Mobilization of Self-Inactivating (SIN) Lentiviral Vectors Containing Globin Regulatory Elements Compared to Those Containing a Retroviral Long Terminal Repeat.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5271-5271
Author(s):  
Hideki Hanawa ◽  
Derek A. Persons ◽  
Takashi Shimada ◽  
Arthur W. Nienhuis
Keyword(s):  
Stem Cell ◽  
Long Terminal Repeat ◽  
Hela Cells ◽  
Lentiviral Vector ◽  
Severe Combined Immunodeficiency ◽  
Lentiviral Vectors ◽  
Regulatory Elements ◽  
Terminal Repeat ◽  
Vector Genome ◽  
The Common

Abstract Stem cell transfer was successful in treating severe combined immunodeficiency due to deficiencies of the common γ-chain of the lymphoid cytokine receptor (N Engl J Med346:1185, 2002) and adenosine deaminase (Science296:2410, 2002) but 2 of 10 children in the common γ-chain trial developed a lymphoproliferative disease secondary in part due to activation of the LMO2 proto-oncogene by the retroviral long terminal repeat (LTR) (Science302:415, 2003). Both oncoretroviral and lentiviral vectors integrate preferentially into transcriptionally active genes so that vector design to improve safety is important. In focusing on the interactions between vector sequences and the genome surrounding integration sites, we have used self-inactivating (SIN) lentiviral vectors with transcriptionally inactive LTRs. One assay detects mobilization of the vector genome by rescue of a GFP marker while a second detects vector encoded tat transcription (Blood102:249a, 2003). Approximately 1 in 1,000 to 1 in 3,000 vector genomes containing the Mouse Stem Cell Virus (MSCV) LTR are transcribed with transcription arising from cryptic promoters within the vector genome or from nearby genomic sequences. The frequency of genome transcription was diminished by removal of the MSCV LTR enhancer or by addition of an insulator element from the β-globin locus control region (LCR) to the lentiviral SIN LTR. We have now evaluated the effect of globin regulatory elements on transcription of integrated SIN lentiviral vector genomes. Initially, we substituted the LTR GFP cassette in vector MSCV-U3 for one in which elements from the β-globin LCR were linked to the β-globin promoter driving GFP in the reverse transcriptional orientation (d432βGFPim). Two additional vectors were also studied; the globin LCR β-promoter GFP cassette was reversed and the globin RNA processing signals removed to derive vector Fd432βGFP and then it was modified by substituting larger LCR elements to derive vector FmLARβV5GFP. Vector preparations made by co-transfection of 293T cells with vector and packaging plasmids had transducing titers from 1.8 x 107 to 3.0 x 107 TU/ml as assayed on HeLa cells. Three separate polyclonal 293T cell populations transduced 3 times at high vector concentration had copy numbers of the SIN-proviral genomes, as measured by RealTime PCR, that averaged 41 with a range of 23–68. Vector mobilization was evaluated by transfection of these populations with packaging plasmids. Conditioned media harvested from the transfected cells were then assayed for transfer of the GFP marker on HeLa cells. The mobilized titers were normalized based on vector copy number. The mobilized titer of the MSCV-U3 was 38,000 ± 3500 and that of d432βGFPim was only 810 ± 100 (p = 0.0004). The mobilized titers of the vectors in which the globin regulatory elements were in the forward orientation were also significantly lower than MSCV-U3; Fd432βGFP was 2100 ± 250 (p = 0.005) and FmLARβV5GFP was 1600 ± 120 (p = 0.0005). Those data suggest that the globin LCR elements are less likely than the retroviral LTR to induce transcription of the integrated vector genome in nonerythroid cells. Our results combined with ongoing analysis of the influence of vector integration on expression of surrounding genes in separate studies will provide a safety profile of globin lentiviral vectors to guide the development of future clinical protocols.

scholarly journals In vivo targeting of lentiviral vectors pseudotyped with the Tupaia paramyxovirus H glycoprotein bearing a cell-specific ligand

2021 ◽  
Author(s):  
Takele Argaw ◽  
Michael P. Marino ◽  
Andrew Timmons ◽  
Lindsey Eldridge ◽  
Kazuyo Takeda ◽  
...  
Keyword(s):  
Lentiviral Vectors ◽  
A Cell ◽  
Specific Ligand

scholarly journals MacroH2A histone variants modulate enhancer activity to repress oncogenic programs and cellular reprogramming

2021 ◽  
Author(s):  
Alexandre Gaspar-Maia ◽  
Wazim Mohammed Ismail ◽  
Amelia Mazzone ◽  
Jagneet Kaur ◽  
Stephanie Safgren ◽  
...  
Keyword(s):  
Regulatory Elements ◽  
Histone Variants ◽  
Cellular Reprogramming ◽  
Negative Regulator ◽  
Cellular Heterogeneity ◽  
Enhancer Activity ◽  
Active Enhancer ◽  
A Cell ◽  
Cell Type Specific

Abstract Considerable efforts have been made to characterize active enhancer elements, which can be annotated by accessible chromatin and H3 lysine 27 acetylation (H3K27ac). However, apart from poised enhancers that are observed in early stages of development and putative silencers, the functional significance of cis-regulatory elements lacking H3K27ac is poorly understood. Here we show that macroH2A histone variants mark a subset of enhancers in normal and cancer cells, which we coined ‘macroH2A-Bound Enhancers’, that negatively modulate enhancer activity. We find macroH2A variants enriched at enhancer elements that are devoid of H3K27ac in a cell type-specific manner, indicating a role for macroH2A at inactive enhancers to maintain cell identity. In following, reactivation of macro-bound enhancers is associated with oncogenic programs in breast cancer and its repressive role is correlated with the activity of macroH2A2 as a negative regulator of BRD4 chromatin occupancy. Finally, through single cell epigenomic profiling, we show that the loss of macroH2A2 leads to increased cellular heterogeneity that may help to explain the role of macroH2A variants in defining oncogenic transcriptional dependencies.

scholarly journals Auxin/AID versus conventional knockouts: distinguishing the roles of CENP-T/W in mitotic kinetochore assembly and stability

Open Biology ◽  
2016 ◽  
Vol 6 (1) ◽  
pp. 150230 ◽  
Author(s):  
Laura Wood ◽  
Daniel G. Booth ◽  
Giulia Vargiu ◽  
Shinya Ohta ◽  
Flavia deLima Alves ◽  
...  
Keyword(s):  
Protein Function ◽  
Mitotic Chromosomes ◽  
Protein Levels ◽  
Kinetochore Assembly ◽  
Rnai Technology ◽  
Gradual Loss ◽  
A Cell ◽  
Stable Association ◽  
Or Gene ◽  
Regulated Gene Expression

Most studies using knockout technologies to examine protein function have relied either on shutting off transcription (conventional conditional knockouts with tetracycline-regulated gene expression or gene disruption) or destroying the mature mRNA (RNAi technology). In both cases, the target protein is lost at a rate determined by its intrinsic half-life. Thus, protein levels typically fall over at least 1–3 days, and cells continue to cycle while exposed to a decreasing concentration of the protein. Here we characterise the kinetochore proteome of mitotic chromosomes isolated from a cell line in which the essential kinetochore protein CENP-T is present as an auxin-inducible degron (AID) fusion protein that is fully functional and able to support the viability of the cells. Stripping of the protein from chromosomes in early mitosis via targeted proteasomal degradation reveals the dependency of other proteins on CENP-T for their maintenance in kinetochores. We compare these results with the kinetochore proteome of conventional CENP-T/W knockouts. As the cell cycle is mostly formed from G1, S and G2 phases a gradual loss of CENP-T/W levels is more likely to reflect dependencies associated with kinetochore assembly pre-mitosis and upon entry into mitosis. Interestingly, a putative super-complex involving Rod-Zw10-zwilch (RZZ complex), Spindly, Mad1/Mad2 and CENP-E requires the function of CENP-T/W during kinetochore assembly for its stable association with the outer kinetochore, but once assembled remains associated with chromosomes after stripping of CENP-T during mitosis. This study highlights the different roles core kinetochore components may play in the assembly of kinetochores (upon entry into mitosis) versus the maintenance of specific components (during mitosis).

Non-genic inheritance of cellular handedness

Development ◽  
1989 ◽  
Vol 105 (3) ◽  
pp. 447-456 ◽  
Author(s):  
E.M. Nelsen ◽  
J. Frankel ◽  
L.M. Jenkins
Keyword(s):  
Positional Information ◽  
Regulatory Elements ◽  
Genetic Change ◽  
Surface Structures ◽  
Phenotypic Alteration ◽  
Left Handed ◽  
A Cell ◽  
Usual Order ◽  
Genic Control ◽  
Rule Out

Ciliates exhibit an asymmetry in arrangement of surface structures around the cell which could be termed handedness. If the usual order of placement of structures defines a ‘right-handed’ (RH) cell, then a cell with this order reversed would be ‘left-handed’ (LH). Such LH forms appear to be produced in Tetrahymena thermophila through aberrant reorganization of homopolar doublets back to the singlet condition. Four clones of LH forms were selected and subjected to genetic analysis to test whether this drastic phenotypic alteration resulted from a nuclear genetic change. The results of this analysis indicate that the change in handedness is not due to a genetic change in either the micronucleus or macronucleus. The LH form can, under certain circumstances, revert to the RH form, but typically it propagates itself across both vegetative and sexual generations with similar fidelity. While this analysis does not formally rule out certain possibilities of nuclear genic control involving regulatory elements transmitted through the cytoplasm, when the circumstances of origin and propagation of the LH condition are taken into account direct cortical perpetuation seems far more likely. Here we outline a conceptual framework centred on the idea of longitudinally propagated positional information; the positive evidence supporting this idea as well as further application of the idea itself are presented in the accompanying paper.

scholarly journals Interaction between two different regulatory elements activates the murine alpha A-crystallin gene promoter in explanted lens epithelia.

1987 ◽  
Vol 7 (5) ◽  
pp. 1807-1814 ◽  
Author(s):  
A B Chepelinsky ◽  
B Sommer ◽  
J Piatigorsky
Keyword(s):  
Dna Sequences ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Regulatory Sequences ◽  
Base Pairs ◽  
Hybrid Gene ◽  
Promoter Sequences ◽  
Hybrid Genes ◽  
Cat Gene ◽  
Cat Expression

Previous experiments have indicated that 5' flanking DNA sequences (nucleotides-366 to +46) are capable of regulating the lens-specific transcription of the murine alpha A-crystallin gene. Here we have analyzed these 5' regulatory sequences by transfecting explanted embryonic chicken lens epithelia with different alpha A-crystallin-CAT (chloramphenicol acetyltransferase) hybrid genes (alpha A-crystallin promoter sequences fused to the bacterial CAT gene in the pSVO-CAT expression vector). The results indicated the presence of a proximal (-88 to +46) and a distal (-111 to -88) domain which must interact for promoter function. Deletion experiments showed that the sequence between -88 and -60 was essential for function of the proximal domain in the explanted epithelia. A synthetic oligonucleotide containing the sequence between -111 and -84 activated the proximal domain when placed in either orientation 57 base pairs upstream from position -88 of the alpha A-crystallin-CAT hybrid gene.

Transfection of calcitonin gene regulatory elements into a cell culture model of the C cell

1990 ◽  
Vol 5 (2) ◽  
pp. 165-171 ◽  
Author(s):  
Gilbert J. Cote ◽  
Ronald V. Abruzzese ◽  
Robert F. Gagel ◽  
CEES J. M. Lips
Keyword(s):  
Cell Culture ◽  
Regulatory Elements ◽  
Cell Culture Model ◽  
C Cell ◽  
Culture Model ◽  
Calcitonin Gene ◽  
Gene Regulatory Elements ◽  
A Cell ◽  
Gene Regulatory

scholarly journals Fungal α-arabinofuranosidases of glycosyl hydrolase families 51 and 54 show a dual arabinofuranosyl- and galactofuranosyl-hydrolyzing activity

2012 ◽  
Vol 393 (8) ◽  
pp. 767-775 ◽  
Author(s):  
Boris Tefsen ◽  
Ellen L. Lagendijk ◽  
Joohae Park ◽  
Michiel Akeroyd ◽  
Doreen Schachtschabel ◽  
...  
Keyword(s):  
Cell Wall ◽  
Enzyme Activity ◽  
Molecular Modeling ◽  
Aspergillus Niger ◽  
Glycosyl Hydrolase ◽  
Hydrolase Activity ◽  
Gene Encoding ◽  
A Cell ◽  
Or Gene

Abstract Aspergillus niger possesses a galactofuranosidase activity, however, the corresponding enzyme or gene encoding this enzyme has never been identified. As evidence is mounting that enzymes exist with affinity for both arabinofuranose and galactofuranose, we investigated the possibility that α-l-arabinofuranosidases, encoded by the abfA and abfB genes, are responsible for the galactofuranosidase activity of A. niger. Characterization of the recombinant AbfA and AbfB proteins revealed that both enzymes do not only hydrolyze p-nitrophenyl-α-l-arabinofuranoside (pNp-α-Araf) but are also capable of hydrolyzing p-nitrophenyl-β-d-galactofuranoside (pNp-β-Galf). Molecular modeling of the AbfB protein with pNp-β-Galf confirmed the possibility for AbfB to interact with this substrate, similarly as with pNp-α-Araf. We also show that galactomannan, a cell wall compound of A. niger, containing β-linked terminal and internal galactofuranosyl moieties, can be degraded by an enzyme activity that is present in the supernatant of inulin-grown A. niger. Interestingly, purified AbfA and AbfB did not show this hydrolyzing activity toward A. nigergalactomannan. In summary, our studies demonstrate that AbfA and AbfB, α-l-arabinofuranosidases from different families, both contain a galactofuranose (Galf)-hydrolyzing activity. In addition, our data support the presence of a Galf-hydrolase activity expressed by A. niger that is capable of degrading fungal galactomannan.

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