Hox-2.3 upstream sequences mediate lacZ expression in intermediate mesoderm derivatives of transgenic mice

Development ◽  
1990 ◽  
Vol 109 (4) ◽  
pp. 775-786 ◽  
Author(s):  
C. Kress ◽  
R. Vogels ◽  
W. De Graaff ◽  
C. Bonnerot ◽  
F. Meijlink ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Regulatory Elements ◽  
Lacz Expression ◽  
Lateral Plate ◽  
Intermediate Mesoderm ◽  
Intergenic Sequences ◽  
Mesonephric Duct ◽  
Metanephric Kidney ◽  
Beta Galactosidase

The mouse Hox-2.3 gene contains an Antp-like homeobox sequence and is expressed in a spatially restricted anteroposterior domain during development. To study the molecular basis of this differential gene regulation, we set out to characterize the cis-regulatory elements mediating Hox-2.3 expression during embryogenesis. We show that a fragment extending 1316 base pairs (bp) upstream of the transcription start site, thus corresponding to the Hox-2.4/Hox-2.3 intergenic sequences is capable of mediating luciferase gene transcription in transfected cells in vitro and lacZ expression in transgenic mice. The beta-galactosidase-staining pattern in embryos was found to be strikingly similar to the Hox-2.3 in situ hybridization pattern in intermediate mesoderm derivatives: high levels of both Hox-2.3 transcripts and beta-galactosidase activity were found in the mesonephric duct-derived epithelium of the meso- and metanephric kidney and associated ducts, from the time these structures first appeared on throughout development. The transgene apparently lacks sequences needed for correct Hox-2.3 expression in somitic and lateral plate mesoderm and in neurectoderm. These results document the involvement of distinct regulatory elements in Hox gene expression in subsets of cells with distinct developmental fate, situated at similar positions along the anteroposterior axis of the embryo.

scholarly journals Two Pax-binding sites are required for early embryonic brain expression of an Engrailed-2 transgene

Development ◽  
1996 ◽  
Vol 122 (2) ◽  
pp. 627-635 ◽  
Author(s):  
D.L. Song ◽  
G. Chalepakis ◽  
P. Gruss ◽  
A.L. Joyner
Keyword(s):  
Dna Binding ◽  
Transgenic Mice ◽  
Binding Sites ◽  
Regulatory Elements ◽  
Developing Brain ◽  
Molecular Evidence ◽  
Regulatory Sequences ◽  
Pax Genes

The temporally and spatially restricted expression of the mouse Engrailed (En) genes is essential for development of the midbrain and cerebellum. The regulation of En-2 expression was studied using in vitro protein-DNA binding assays and in vivo expression analysis in transgenic mice to gain insight into the genetic events that lead to regionalization of the developing brain. A minimum En-2 1.0 kb enhancer fragment was defined and found to contain multiple positive and negative regulatory elements that function in concert to establish the early embryonic mid-hindbrain expression. Furthermore, the mid-hindbrain regulatory sequences were shown to be structurally and functionally conserved in humans. The mouse paired-box-containing genes Pax-2, Pax-5 and Pax-8 show overlapping expression with the En genes in the developing brain. Significantly, two DNA-binding sites for Pax-2, Pax-5 and Pax-8 proteins were identified in the 1.0 kb En-2 regulatory sequences, and mutation of the binding sites disrupted initiation and maintenance of expression in transgenic mice. These results present strong molecular evidence that the Pax genes are direct upstream regulators of En-2 in the genetic cascade controlling mid-hindbrain development. These mouse studies, taken together with others in Drosophila and zebrafish on the role of Pax genes in controlling expression of En family members, indicate that a Pax-En genetic pathway has been conserved during evolution.

Ectopic Fibroblast Growth Factor Receptor 3 (FGFR3) Expression Mediated by Isotype Switch Recombination.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 78-78 ◽  
Author(s):  
Maurizio Affer ◽  
Davide Robbiani ◽  
Marta Chesi ◽  
Peter L. Bergsagel
Keyword(s):  
Multiple Myeloma ◽  
B Cells ◽  
Transgenic Mice ◽  
Tumor Progression ◽  
Plasma Cells ◽  
Growth Parameters ◽  
Ectopic Expression ◽  
Regulatory Elements ◽  
Igh Locus

Abstract FGFR3 is disregulated in approximately 15% of cases of multiple myeloma (MM) occurring by chromosomal translocation in the Igh switch region which results in the juxtaposition of FGFR3 next to the regulatory elements of the IgH locus, causing its ectopic expression in plasma cells. The acquisition of FGFR3 activating mutations in some tumors indicate a role for FGFR3 in tumor progression. In order to study its role in MM progression we developed a strain of transgenic mice in which the expression of the activated form of FGFR3 is mediated by isotype switch recombination by replacing the IgH gamma1 costant region with an FGFR3-IRES-EGFP cassette in a BAC that covers the entire murine IgH locus. We predict that in the transgenic mice B cells that undergo switch recombination to g1 on the productive allele will also undergo switch recombination to g1 at the transgenic locus and express FGFR3 and EGFP. To evaluate FGFR3 expression in vitro, we collected splenocytes from our transgenic mice and induced their proliferation and differentiation to plasma cells (mainly IgG1) using LPS and IL4. After four days of stimulation, the average number of EGFP positive cells went from 0.2% to 80%. We then analyzed FGFR3 expression by RT PCR, Northern and Western blot and detected LPS/IL4 inducible FGFR3 expression in plasma cells mediated mainly by the Igamma1 promoter and in a subset of them by the Vh promoter. We then tested if forced FGFR3 expression in B cells would affect their proliferation rate and ability to differentiate in vitro by MTT assay, cell cycle analysis and PI staining on LPS/IL4 stimulated splenocytes at different time points. With this system, we did not observe any FGFR3 mediated alteration of splenocytes growth parameters when compared to wild type controls. Similarly, in vivo no monoclonal gammopathy has been observed by serum protein electrophoresis and all the transgenic mice remaine tumor free at 1.5 years. Although we have not been able to demonstrate a tumorigenic role for FGFR3 in switched plasmacells we were able to obtaine regulated FGFR3 expression in them. It is possible that FGFR3 expression alone is not sufficient to induce plasma cells transformation and that FGFR3 has a dispensable role in tumor initiation, but that it could still play a role in tumor progression. Consistently, FGFR3 expression is lost in about 25% of multiple myeloma wih a t(4;14). To address this hypotesis we are currently crossing these mice with MMSET (the other gene disregulated in the t(4;14)) transgenic mice to investigate a possible collaborative role between these two genes in the development of MM.

Abstract 463: The Key Regulatory Elements for SM22 Transcription

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Maozhou Yang ◽  
Soren Warming ◽  
Jianbin Shen ◽  
Hong Jiang ◽  
Hui J Li ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Transgenic Mice ◽  
Molecular Mechanisms ◽  
Expression Patterns ◽  
Vascular Diseases ◽  
Regulatory Elements ◽  
Cardiovascular Development ◽  
Lacz Expression ◽  
Carg Box

Gene transcription is controlled by an array of transcriptional regulatory elements. SM22 gene regulation has been widely used to characterize the molecular mechanisms of smooth muscle cell (SMC) phenotypic modulation during cardiovascular development and in vascular diseases. Our previous studies show that the proximal CArG box (CArGnear) in the SM22 promoter is required for its transcription in arterial smooth muscle cells (SMC). However, the role of the CArG box in visceral SMCs has not yet been explored. Here we aim to determine the role of CArG boxes in regulating S M22 transcription in both vascular and visceral SMCs. Using bacterial chromosome (BAC) recombineering, we knock-in a lacZ reporter into a SM22 BAC to trace SM22 transcriptional activities in transgenic mice. Anatomic/histology analyses show that the lacZ expression patterns in the BAC transgenic mice recapitulate that of the endogenous SM22 transcription during embryogenesis and in adult . Similar to the endogenous SM22 regulation, the expression of lacZ is highly sensitive to vascular remodeling: carotid injury abolishes lacZ expression in the arterial wall. Using seamless BAC recombineering mutagenesis, we generate mutations in the proximal and/or distal CArG box in the SM22-lacZ-BAC. Consistent with our previous results, we find that mutating the CArGnear box disrupts the lacZ expression in the aorta; this mutation also drastically reduces its expression in visceral SMCs including stomach, uterus and bladder. Interestingly, mutating the distal CArG (CArGfar) box does not affect the lacZ expression in arterial, venous and visceral SMCs. Mutating both CArG boxes nearly abolishes lacZ expression in all SMCs. This study provides evidence supporting the generation of SM22 knockout mice by mutating the CArG boxes in the SM22 promoter using the CRISPR technology.

Desmin sequence elements regulating skeletal muscle-specific expression in transgenic mice

Development ◽  
1993 ◽  
Vol 117 (3) ◽  
pp. 947-959 ◽  
Author(s):  
Z. Li ◽  
P. Marchand ◽  
J. Humbert ◽  
C. Babinet ◽  
D. Paulin
Keyword(s):  
Transgenic Mice ◽  
Reporter Gene ◽  
Skeletal Muscles ◽  
Regulatory Elements ◽  
Limb Bud ◽  
Regulatory Sequence ◽  
Cardiac Muscle Cells ◽  
Specific Expression ◽  
Foetal Development ◽  
Beta Galactosidase

During the development of the mouse embryo, desmin is one of the first muscle proteins detected in both the heart and the somites. The expression of the desmin gene differs from most other muscle genes, since it is initiated in replicating myoblasts and accumulates as the muscle differentiates. We have characterized a muscle-specific enhancer which directs the expression of desmin in vitro in the myoblasts and myotubes of C2 cells but not in non-myogenic cells. We report here on the generation and characterization of transgenic mice bearing a transgene in which the 1 kb DNA 5′ regulatory sequence of the desmin gene is linked to a reporter gene coding for Escherichia coli beta-galactosidase (Des1-nlacZ). The enhancer activity of the desmin promoter is very strong and the reporter gene expression is easily detected in tissue sections. We have demonstrated that the regulatory elements present in the transgene Des1-nlacZ are sufficient to direct muscle-specific and developmentally regulated expression of nlacZ in skeletal muscles. Endogenous desmin expression and transgene activity were found to be correlated during the development of skeletal muscles. The transgene was expressed in the committed mononucleate myoblasts as well as in the myotubes. In addition, we have shown that the desmin-derived sequences direct a highly selective expression of nlacZ in cells that leave the somites and invade the limb bud, indicating that the cells that migrate from the somites are already predetermined for myogenesis. In contrast, smooth and cardiac muscle cells were beta-galactosidase negative both during embryonic and foetal development. Interestingly, the transgene was found to be expressed in the conduction system of the heart, which exhibits many features characteristic of skeletal muscles.

Synthesis of a Novel Curcumin Derivative as a Potential Imaging Probe in Alzheimer’s Disease Imaging

2019 ◽  
Vol 16 (8) ◽  
pp. 723-731 ◽  
Author(s):  
Alexander Sturzu ◽  
Sumbla Sheikh ◽  
Hubert Kalbacher ◽  
Thomas Nägele ◽  
Christopher Weidenmaier ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Flow Cytometry ◽  
Transgenic Mice ◽  
Ex Vivo ◽  
Amyloid Plaques ◽  
Laser Scanning Microscopy ◽  
Β Amyloid ◽  
Curcumin Derivative

Background: Curcumin has been of interest in the field of Alzheimer’s disease. Early studies on transgenic mice showed promising results in the reduction of amyloid plaques.However, curcumin is very poorly soluble in aqueous solutions and not easily accessible to coupling as it contains only phenolic groups as potential coupling sites. For these reasons only few imaging studies using curcumin bound as an ester were performed and curcumin is mainly used as nutritional supplement. Methods: In the present study we produced an aminoethyl ether derivative of curcumin using a nucleophilic substitution reaction. This is a small modification and should not impact the properties of curcumin while introducing an easily accessible reactive amino group. This novel compound could be used to couple curcumin to other molecules using the standard methods of peptide synthesis. We studied the aminoethyl-curcumin compound and a tripeptide carrying this aminoethyl-curcumin and the fluorescent dye fluorescein (FITC-curcumin) in vitro on cell culture using confocal laser scanning microscopy and flow cytometry. Then these two substances were tested ex vivo on brain sections prepared from transgenic mice depicting Alzheimer-like β-amyloid plaques. Results: In the in vitro CLSM microscopy and flow cytometry experiments we found dot-like unspecific uptake and only slight cytotoxicity correlating with this uptake. As these measurements were optimized for the use of fluorescein as dye we found that the curcumin at 488nm fluorescence excitation was not strong enough to use it as a fluorescence marker in these applications. In the ex vivo sections CLSM experiments both the aminoethyl-curcumin and the FITC-curcumin peptide bound specifically to β- amyloid plaques. Conclusion: In conclusion we successfully produced a novel curcumin derivative which could easily be coupled to other imaging or therapeutic molecules as a sensor for amyloid plaques.

scholarly journals DNA-directed in vitro synthesis of beta-galactosidase. Studies with purified factors.

1977 ◽  
Vol 252 (19) ◽  
pp. 6889-6894 ◽  
Author(s):  
H F Kung ◽  
B Redfield ◽  
B V Treadwell ◽  
B Eskin ◽  
C Spears ◽  
...  
Keyword(s):  
In Vitro Synthesis ◽  
Beta Galactosidase

scholarly journals Reversal of senescence-associated beta-galactosidase expression during in vitro three-dimensional tissue-engineering of human chondrocytes in a polymer scaffold

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shojiro Katoh ◽  
Atsuki Fujimaru ◽  
Masaru Iwasaki ◽  
Hiroshi Yoshioka ◽  
Rajappa Senthilkumar ◽  
...  
Keyword(s):  
Regenerative Medicine ◽  
Replicative Senescence ◽  
Cultured Cells ◽  
Three Dimensional ◽  
Human Chondrocytes ◽  
Cartilage Tissue ◽  
Optimal Method ◽  
Beta Galactosidase

AbstractRegenerative medicine applications require cells that are not inflicted with senescence after in vitro culture for an optimal in vivo outcome. Methods to overcome replicative senescence include genomic modifications which have their own disadvantages. We have evaluated a three-dimensional (3D) thermo-reversible gelation polymer (TGP) matrix environment for its capabilities to reverse cellular senescence. The expression of senescence-associated beta-galactosidase (SA-βgal) by human chondrocytes from osteoarthritis-affected cartilage tissue, grown in a conventional two-dimensional (2D) monolayer culture versus in 3D-TGP were compared. In 2D, the cells de-differentiated into fibroblasts, expressed higher SA-βgal and started degenerating at 25 days. SA-βgal levels decreased when the chondrocytes were transferred from the 2D to the 3D-TGP culture, with cells exhibiting a tissue-like growth until 42–45 days. Other senescence associated markers such as p16INK4a and p21 were also expressed only in 2D cultured cells but not in 3D-TGP tissue engineered cartilage. This is a first-of-its-kind report of a chemically synthesized and reproducible in vitro environment yielding an advantageous reversal of aging of human chondrocytes without any genomic modifications. The method is worth consideration as an optimal method for growing cells for regenerative medicine applications.

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