Scaffold attachment region–containing retrovirus vectors improve long-term proviral expression after transplantation of GFP-modified CD34+ baboon repopulating cells

AbstractSustained high-level proviral expression is important for clinical applications of gene therapy. Genetic elements including the β-interferon scaffold attachment region (SAR) have been shown to improve transgene expression in hematopoietic cells. We hypothesized that SAR elements might improve expression and allow the preselection of successfully transduced cells. Thus, we transplanted green fluorescent protein (GFP)–selected cells, half of which had been transduced with either SAR or non–SAR-containing retrovirus vectors, into 3 animals. All animals showed delayed engraftment compared with historic controls (28 vs 15.5 days). GFP marking was seen at levels up to 8% but declined over the first 6 weeks. Importantly, fluorescence intensity was 2- to 9-fold increased in progeny of SAR versus non–SAR vector–modified cells in all hematopoietic lineages for the duration of follow-up (6-12 months). In conclusion, the use of SAR-containing vectors improved transgene expression in hematopoietic repopulating cells, which may obviate the need for multicopy integration to achieve high-level expression and reduce the risk for insertional mutagenesis.

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The β-Interferon Scaffold Attachment Region Confers High-Level Transgene Expression and Avoids Extinction by Epigenetic Modifications of Integrated Provirus in Adipose Tissue-Derived Human Mesenchymal Stem Cells

Tissue Engineering Part C Methods ◽

10.1089/ten.tec.2010.0383 ◽

2011 ◽

Vol 17 (3) ◽

pp. 275-287 ◽

Cited By ~ 11

Author(s):

Rafael Moreno ◽

Itziar Martínez ◽

Jordi Petriz ◽

Marga Nadal ◽

Xavier Tintoré ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Adipose Tissue ◽

Transgene Expression ◽

Human Mesenchymal Stem Cells ◽

Epigenetic Modifications ◽

Attachment Region ◽

Scaffold Attachment Region ◽

High Level

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Multicopy Integration of Heterologous Genes, Using the Lactococcal Group II Intron Targeted to Bacterial Insertion Sequences

Applied and Environmental Microbiology ◽

10.1128/aem.02992-05 ◽

2006 ◽

Vol 72 (9) ◽

pp. 6088-6093 ◽

Cited By ~ 19

Author(s):

Helen Rawsthorne ◽

Kevin N. Turner ◽

David A. Mills

Keyword(s):

Fluorescent Protein ◽

Group Ii Intron ◽

Multicopy Plasmid ◽

Bacterial Genomes ◽

Gfp Expression ◽

High Frequencies ◽

Multicopy Integration ◽

Group Ii ◽

Green Fluorescent ◽

High Level

ABSTRACT Group II introns are mobile genetic elements that can be redirected to invade specific genes. Here we describe the use of the lactococcal group II intron, Ll.ltrB, to achieve multicopy delivery of heterologous genes into the genome of Lactococcus lactis IL1403-UCD without the need for selectable markers. Ll.ltrB was retargeted to invade three transposase genes, the tra gene found in IS904 (tra904), tra981, and tra983, of which 9, 10, and 14 copies, respectively, were present in IL1403-UCD. Intron invasion of tra904, tra981, and tra983 allele groups occurred at high frequencies, and individual segregants possessed anywhere from one to nine copies of intron in the respective tra alleles. To achieve multicopy delivery of a heterologous gene, a green fluorescent protein (GFP) marker was cloned into the tra904-targeted Ll.ltrB, and the resultant intron (Ll.ltrB::GFP) was induced to invade the L. lactis tra904 alleles. Segregants possessing Ll.ltrB::GFP in three, four, five, six, seven, and eight copies in different tra904 alleles were obtained. In general, increasing the chromosomal copy number of Ll.ltrB::GFP resulted in strains expressing successively higher levels of GFP. However, strains possessing the same number of Ll.ltrB::GFP copies within different sets of tra904 alleles exhibited differential GFP expression, and segregants possessing seven or eight copies of Ll.ltrB::GFP grew poorly upon induction, suggesting that GFP expression from certain combinations of alleles was detrimental. The highest level of GFP expression was observed from a specific six-copy variant that produced GFP at a level analogous to that obtained with a multicopy plasmid. In addition, the high level of GFP expression was stable for over 120 generations. This work demonstrates that stable multicopy integration of heterologous genes can be readily achieved in bacterial genomes with group II intron delivery by targeting repeated elements.

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Multicopy Integration and Expression of Heterologous Genes in Methylobacterium extorquens ATCC 55366

Applied and Environmental Microbiology ◽

10.1128/aem.72.1.753-759.2006 ◽

2006 ◽

Vol 72 (1) ◽

pp. 753-759 ◽

Cited By ~ 22

Author(s):

Young J. Choi ◽

Denis Bourque ◽

Lyne Morel ◽

Denis Groleau ◽

Carlos B. Míguez

Keyword(s):

Green Fluorescent Protein ◽

Target Genes ◽

Fluorescent Protein ◽

Expression System ◽

Attachment Site ◽

Methylobacterium Extorquens ◽

Gene Copies ◽

Multicopy Integration ◽

Green Fluorescent ◽

High Level

ABSTRACT High-level expression of chromosomally integrated genes in Methylobacterium extorquens ATCC 55366 was achieved under the control of the strong M. extorquens AM1 methanol dehydrogenase promoter (P mxaF ) using the mini-Tn7 transposon system. Stable maintenance and expression of the integrated genes were obtained in the absence of antibiotic selective pressure. Furthermore, using this technology, a multicopy integration protocol for M. extorquens was also developed. Chromosomal integration of one to five copies of the gene encoding the green fluorescent protein (gfp) was achieved. The multicopy-based expression system permitted expression of a preset number of gene copies. A unique specific Tn7 integration locus in the chromosome of M. extorquens, known as the Tn7 attachment site (attTn7 site), was identified. This single attTn7 site was identified in an intergenic region between glmS, which encodes the essential enzyme glucosamine-6-phosphate synthetase, and dhaT, which encodes 1,3-propanediol dehydrogenase. The fact that the integration event is site specific and the fact that the attTn7 site is a noncoding region of the chromosome make the mini-Tn7 transposon system very useful for insertion of target genes and subsequent expression. In all transformants tested, expression and segregation of the transforming gene were stable without generation of secondary mutations in the host. In this paper, we describe single and multicopy chromosome integration and stable expression of heterologous genes (bgl [β-galactosidase], est [esterase], and gfp [green fluorescent protein]) in M. extorquens.

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High-Level Transgene Expression in Plant Cells: Effects of a Strong Scaffold Attachment Region from Tobacco

The Plant Cell ◽

10.2307/3870291 ◽

1996 ◽

Vol 8 (5) ◽

pp. 899 ◽

Cited By ~ 1

Author(s):

George C. Allen ◽

Gerald Hall ◽

Susan Michalowski ◽

Winnell Newman ◽

Steven Spiker ◽

...

Keyword(s):

Transgene Expression ◽

Plant Cells ◽

Attachment Region ◽

Scaffold Attachment Region ◽

High Level

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Neogenesis of cerebellar Purkinje neurons from gene-marked bone marrow cells in vivo

The Journal of Cell Biology ◽

10.1083/jcb.200105103 ◽

2001 ◽

Vol 155 (5) ◽

pp. 733-738 ◽

Cited By ~ 181

Author(s):

Josef Priller ◽

Derek A. Persons ◽

Francisco F. Klett ◽

Gerd Kempermann ◽

Georg W. Kreutzberg ◽

...

Keyword(s):

Bone Marrow ◽

Purkinje Cells ◽

Fluorescent Protein ◽

Bone Marrow Cells ◽

Purkinje Neurons ◽

Acid Decarboxylase ◽

Green Fluorescent ◽

High Level

The versatility of stem cells has only recently been fully recognized. There is evidence that upon adoptive bone marrow (BM) transplantation (BMT), donor-derived cells can give rise to neuronal phenotypes in the brains of recipient mice. Yet only few cells with the characteristic shape of neurons were detected 1–6 mo post-BMT using transgenic or newborn mutant mice. To evaluate the potential of BM to generate mature neurons in adult C57BL/6 mice, we transferred the enhanced green fluorescent protein (GFP) gene into BM cells using a murine stem cell virus-based retroviral vector. Stable and high level long-term GFP expression was observed in mice transplanted with the transduced BM. Engraftment of GFP-expressing cells in the brain was monitored by intravital microscopy. In a long-term follow up of 15 mo post-BMT, fully developed Purkinje neurons were found to express GFP in both cerebellar hemispheres and in all chimeric mice. GFP-positive Purkinje cells were also detected in BM chimeras from transgenic mice that ubiquitously express GFP. Based on morphologic criteria and the expression of glutamic acid decarboxylase, the newly generated Purkinje cells were functional.

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High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco.

The Plant Cell ◽

10.1105/tpc.8.5.899 ◽

1996 ◽

Vol 8 (5) ◽

pp. 899-913 ◽

Cited By ~ 110

Author(s):

G C Allen ◽

G Hall ◽

S Michalowski ◽

W Newman ◽

S Spiker ◽

...

Keyword(s):

Transgene Expression ◽

Plant Cells ◽

Attachment Region ◽

Scaffold Attachment Region ◽

High Level

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Multiciliated cell basal bodies align in stereotypical patterns coordinated by the apical cytoskeleton

The Journal of Cell Biology ◽

10.1083/jcb.201601023 ◽

2016 ◽

Vol 214 (5) ◽

pp. 571-586 ◽

Cited By ~ 34

Author(s):

Elisa Herawati ◽

Daisuke Taniguchi ◽

Hatsuho Kanoh ◽

Kazuhiro Tateishi ◽

Shuji Ishihara ◽

...

Keyword(s):

Fluorescent Protein ◽

Imaging System ◽

Basal Bodies ◽

Large Numbers ◽

Multiciliated Cells ◽

Alignment Process ◽

Flow Through ◽

Linear Alignment ◽

Green Fluorescent

Multiciliated cells (MCCs) promote fluid flow through coordinated ciliary beating, which requires properly organized basal bodies (BBs). Airway MCCs have large numbers of BBs, which are uniformly oriented and, as we show here, align linearly. The mechanism for BB alignment is unexplored. To study this mechanism, we developed a long-term and high-resolution live-imaging system and used it to observe green fluorescent protein–centrin2–labeled BBs in cultured mouse tracheal MCCs. During MCC differentiation, the BB array adopted four stereotypical patterns, from a clustering “floret” pattern to the linear “alignment.” This alignment process was correlated with BB orientations, revealed by double immunostaining for BBs and their asymmetrically associated basal feet (BF). The BB alignment was disrupted by disturbing apical microtubules with nocodazole and by a BF-depleting Odf2 mutation. We constructed a theoretical model, which indicated that the apical cytoskeleton, acting like a viscoelastic fluid, provides a self-organizing mechanism in tracheal MCCs to align BBs linearly for mucociliary transport.

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Analysis of the Bacillus subtilis spoIIIJ Gene and Its Paralogue Gene, yqjG

Journal of Bacteriology ◽

10.1128/jb.184.7.1998-2004.2002 ◽

2002 ◽

Vol 184 (7) ◽

pp. 1998-2004 ◽

Cited By ~ 47

Author(s):

Takako Murakami ◽

Koki Haga ◽

Michio Takeuchi ◽

Tsutomu Sato

Keyword(s):

Bacillus Subtilis ◽

Membrane Proteins ◽

Vegetative Growth ◽

Fluorescent Protein ◽

Specific Expression ◽

Rich Media ◽

Green Fluorescent ◽

High Level ◽

Fluorescent Protein Reporter

ABSTRACT The Bacillus subtilis spoIIIJ gene, which has been proven to be vegetatively expressed, has also been implicated as a sporulation gene. Recent genome sequencing information in many organisms reveals that spoIIIJ and its paralogous gene, yqjG, are conserved from prokaryotes to humans. A homologue of SpoIIIJ/YqjG, the Escherichia coli YidC is involved in the insertion of membrane proteins into the lipid bilayer. On the basis of this similarity, it was proposed that the two homologues act as translocase for the membrane proteins. We studied the requirements for spoIIIJ and yqjG during vegetative growth and sporulation. In rich media, the growth of spoIIIJ and yqjG single mutants were the same as that of the wild type, whereas spoIIIJ yqjG double inactivation was lethal, indicating that together these B. subtilis translocase homologues play an important role in maintaining the viability of the cell. This result also suggests that SpoIIIJ and YqjG probably control significantly overlapping functions during vegetative growth. spoIIIJ mutations have already been established to block sporulation at stage III. In contrast, disruption of yqjG did not interfere with sporulation. We further show that high level expression of spoIIIJ during vegetative phase is dispensable for spore formation, but the sporulation-specific expression of spoIIIJ is necessary for efficient sporulation even at the basal level. Using green fluorescent protein reporter to monitor SpoIIIJ and YqjG localization, we found that the proteins localize at the cell membrane in vegetative cells and at the polar and engulfment septa in sporulating cells. This localization of SpoIIIJ at the sporulation-specific septa may be important for the role of spoIIIJ during sporulation.

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GluA1 trafficking and metabotropic NMDA: addressing results from other laboratories inconsistent with ours

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2013.0145 ◽

2014 ◽

Vol 369 (1633) ◽

pp. 20130145 ◽

Cited By ~ 21

Author(s):

Sadegh Nabavi ◽

Rocky Fox ◽

Stephanie Alfonso ◽

Jonathan Aow ◽

Roberto Malinow

Keyword(s):

Fluorescent Protein ◽

Long Term Potentiation ◽

Long Term Depression ◽

Mk 801 ◽

Over Expression ◽

Term Potentiation ◽

The Difference ◽

Green Fluorescent ◽

Gfp Tag

We have previously shown that when over-expressed in neurons, green fluorescent protein (GFP) tagged GluA1 (GluA1-GFP) delivery into synapses is dependent on plasticity. A recent study suggests that GluA1 over-expression leads to its incorporation into the synapse, in the absence of additional long-term potentiation-like manipulations. It is possible that a GFP tag was responsible for the difference. Using rectification index as a measure of synaptic delivery of GluA1, we found no difference in the synaptic delivery of GluA1-GFP versus untagged GluA1. We recently published a study showing that while D-APV blocks NMDAr-dependent long-term depression (LTD), MK-801 and 7-chloro kynurenate (7CK) fail to block LTD. We propose a metabotropic function for the NMDA receptor in LTD induction. In contrast to our observations, recent unpublished data suggest that the above antagonists are equally effective in blocking LTD. We noticed different methodology in their study. Here, we show that their methodology has complex effects on synaptic transmission. Therefore, it is not possible to conclude that 7CK is effective in blocking LTD from their type of experiment.

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