scholarly journals A drug-repositioning screen using splicing-sensitive fluorescent reporters identifies novel modulators of VEGF-A splicing with anti-angiogenic properties

Oncogenesis ◽  
2021 ◽  
Vol 10 (5) ◽  
Author(s):  
Eleanor Star ◽  
Megan Stevens ◽  
Clare Gooding ◽  
Christopher W. J. Smith ◽  
Ling Li ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Fluorescent Protein ◽  
Drug Repositioning ◽  
Egfp Expression ◽  
In Vivo Model ◽  
Terminal Exon ◽  
Angiogenic Activity

AbstractAlternative splicing of the vascular endothelial growth factor A (VEGF-A) terminal exon generates two protein families with differing functions. Pro-angiogenic VEGF-Axxxa isoforms are produced via selection of the proximal 3′ splice site of the terminal exon. Use of an alternative distal splice site generates the anti-angiogenic VEGF-Axxxb proteins. A bichromatic splicing-sensitive reporter was designed to mimic VEGF-A alternative splicing and was used as a molecular tool to further investigate this alternative splicing event. Part of VEGF-A’s terminal exon and preceding intron were inserted into a minigene construct followed by the coding sequences for two fluorescent proteins. A different fluorescent protein is expressed depending on which 3′ splice site of the exon is used during splicing (dsRED denotes VEGF-Axxxa and EGFP denotes VEGF-Axxxb). The fluorescent output can be used to follow splicing decisions in vitro and in vivo. Following successful reporter validation in different cell lines and altering splicing using known modulators, a screen was performed using the LOPAC library of small molecules. Alterations to reporter splicing were measured using a fluorescent plate reader to detect dsRED and EGFP expression. Compounds of interest were further validated using flow cytometry and assessed for effects on endogenous VEGF-A alternative splicing at the mRNA and protein level. Ex vivo and in vitro angiogenesis assays were used to demonstrate the anti-angiogenic effect of the compounds. Furthermore, anti-angiogenic activity was investigated in a Matrigel in vivo model. To conclude, we have identified a set of compounds that have anti-angiogenic activity through modulation of VEGF-A terminal exon splicing.

335 CYTOPLASMIC MICROINJECTION OF EXOGENOUS DNA IN IN VITRO AND IN VIVO DERIVED SHEEP EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 263
Author(s):  
F. Pereyra-Bonnet ◽  
A. Gibbons ◽  
M. Cueto ◽  
R. Bevacqua ◽  
L. Escobar ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Genetically Modified ◽  
Fold Increase ◽  
Egfp Expression ◽  
Control Group ◽  
Corpora Lutea ◽  
Exogenous Dna ◽  
Frozen Semen

Microinjection of DNA into the male pronucleus is a commonly used method to generate transgenic animals. However, it is only moderately efficient in several species because it requires proper male pronuclear visualisation, which occurs only in a narrow window of time in mice. The cytoplasmic microinjection of exogenous DNA (eDNA) is an alternative method that has not been fully investigated. Our objective was to evaluate if cytoplasmic microinjection of eDNA is capable of producing genetically modified embryos. In vitro and in vivo derived sheep embryos were cytoplasmically microinjected with pCX-EGFP previously incubated (5 min in a PVP droplet) with oolemma-cytoplasm fragments obtained from donor oocytes by microsurgery. A control group using microinjected plasmid alone was included in the in vivo procedure. For in vitro microinjection, IVF embryos were microinjected with circular plasmid with promoter (50 or 500 ng μL–1) or without promoter (50 ng μL–1) at 6 h after fertilization. The IVF was performed following (Brackett and Olliphant 1975 Biol. Reprod. 12, 260–274) with 15 × 106 spermatozoa mL–1, and presumptive zygotes were cultured in SOF. The expression of enhance green fluorescent protein (EGFP) was determined under blue light. For in vivo microinjection, embryos from superovulated sheep (by standard procedures) were recovered and microinjected with 50 ng μL–1 of linearized plasmid without promoter at 12 h after laparoscopic insemination with frozen semen (100 × 106 spermatozoa per sheep). Plasmid without promoter was used to avoid any possible cytotoxic effect produced by EGFP expression. The microinjection of IVF embryos with 50 ng μL–1 of plasmid was the best condition to produce embryos expressing eDNA (n = 96; 46.9% cleaved; 12.2% blastocysts; 53.0 and 4.1% of green embryos and blastocysts, respectively). Variables between the groups with or without promoter IVF were not statistically different (Fisher test: P < 0.05); however, when 500 ng μL–1 was microinjected, no blastocysts were obtained. In the in vivo embryo production group, 111 presumptive zygotes were microinjected (n = 37; with plasmid alone) from 16 donor sheep (11.5 ± 4.0 corpora lutea; 8.4 ± 4.8 presumptive zygotes recovered; 74.3% recovery rate). The mean time from injection to cleavage was 18.0 ± 4.5 h, and the percentage of cleavage and damage (due to the embryo injection) were >70% and <10%, respectively. Fifty-eight good quality embryos were transferred into the oviducts of 19 surrogate ewes; 12 of them are pregnant (63.1%). The presence of green IVF embryos demonstrates that eDNA was transported to the nucleus after cytoplasmic injection. We believe that the multi-fold increase (50- to 100-fold) in plasmid concentration compared with that used by others was the key step to our successful cytoplasmic microinjection. Accordingly, the new/old methodology described in this study provides an easy DNA construct delivery system of interest for the implementation of early reprogramming events. In addition, results obtained in the near future using in vivo cytoplasmic microinjection with high concentrations of eDNA could revalidate this technique for producing genetically modified large animals.

scholarly journals Multiple cis-acting sequence elements are required for efficient splicing of simian virus 40 small-t antigen pre-mRNA.

1988 ◽  
Vol 8 (9) ◽  
pp. 3582-3590 ◽  
Author(s):  
X Y Fu ◽  
J D Colgan ◽  
J L Manley
Keyword(s):  
Alternative Splicing ◽  
Branch Point ◽  
Splice Site ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Specific Sequence ◽  
Small T Antigen

We have determined the effects of a number of mutations in the small-t antigen mRNA intron on the alternative splicing pattern of the simian virus 40 early transcript. Expansion of the distance separating the small-t pre-mRNA lariat branch point and the shared large T-small t 3' splice site from 18 to 29 nucleotides (nt) resulted in a relative enhancement of small-t splicing in vivo. This finding, coupled with the observation that large-T pre-RNA splicing in vitro was not affected by this expansion, suggests that small-t splicing is specifically constrained by a short branch point-3' splice site distance. Similarly, the distance separating the 5' splice site and branch point (48 nt) was found to be at or near a minimum for small-t splicing, because deletions in this region as small as 2 nt dramatically reduced the ratio of small-t to large-T mRNA that accumulated in transfected cells. Finally, a specific sequence within the small-t intron, encompassing the upstream branch sites used in large-T splicing, was found to be an important element in the cell-specific pattern of early alternative splicing. Substitutions within this region reduced the ratio of small-t to large-T mRNA produced in HeLa cells but had only minor effects in human 293 cells.

Multiple cis-acting sequence elements are required for efficient splicing of simian virus 40 small-t antigen pre-mRNA

1988 ◽  
Vol 8 (9) ◽  
pp. 3582-3590
Author(s):  
X Y Fu ◽  
J D Colgan ◽  
J L Manley
Keyword(s):  
Alternative Splicing ◽  
Branch Point ◽  
Splice Site ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Specific Sequence ◽  
Small T Antigen

We have determined the effects of a number of mutations in the small-t antigen mRNA intron on the alternative splicing pattern of the simian virus 40 early transcript. Expansion of the distance separating the small-t pre-mRNA lariat branch point and the shared large T-small t 3' splice site from 18 to 29 nucleotides (nt) resulted in a relative enhancement of small-t splicing in vivo. This finding, coupled with the observation that large-T pre-RNA splicing in vitro was not affected by this expansion, suggests that small-t splicing is specifically constrained by a short branch point-3' splice site distance. Similarly, the distance separating the 5' splice site and branch point (48 nt) was found to be at or near a minimum for small-t splicing, because deletions in this region as small as 2 nt dramatically reduced the ratio of small-t to large-T mRNA that accumulated in transfected cells. Finally, a specific sequence within the small-t intron, encompassing the upstream branch sites used in large-T splicing, was found to be an important element in the cell-specific pattern of early alternative splicing. Substitutions within this region reduced the ratio of small-t to large-T mRNA produced in HeLa cells but had only minor effects in human 293 cells.

scholarly journals Cx26 keratitis ichthyosis deafness syndrome mutations trigger alternative splicing of Cx26 to prevent expression and cause toxicity in vitro

2019 ◽  
Vol 6 (8) ◽  
pp. 191128 ◽  
Author(s):  
Jonathan Cook ◽  
Elizabeth de Wolf ◽  
Nicholas Dale
Keyword(s):  
Alternative Splicing ◽  
Branch Point ◽  
Splice Site ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Future Research ◽  
Cryptic Splice Site ◽  
Toxicity In Vitro

The Cx26 mRNA has not been reported to undergo alternative splicing. In expressing a series of human keratitis ichthyosis deafness (KID) syndrome mutations of Cx26 (A88V, N14K and A40V), we found the production of a truncated mRNA product. These mutations, although not creating a cryptic splice site, appeared to activate a pre-existing cryptic splice site. The alternative splicing of the mutant Cx26 mRNA could be prevented by mutating the predicted 3′, 5′ splice sites and the branch point. The presence of a C-terminal fluorescent protein tag (mCherry or Clover) was necessary for this alternative splicing to occur. Strangely, Cx26 A88V could cause the alternative splicing of co-expressed WT Cx26—suggesting a trans effect. The alternative splicing of Cx26 A88V caused cell death, and this could be prevented by the 3′, 5′ and branch point mutations. Expression of the KID syndrome mutants could be rescued by combining them with removal of the 5′ splice site. We used this strategy to enable expression of Cx26 A40V-5′ and demonstrate that this KID syndrome mutation removed CO 2 sensitivity from the Cx26 hemichannel. This is the fourth KID syndrome mutation found to abolish the CO 2 -sensitivity of the Cx26 hemichannel, and suggests that the altered CO ­2 -sensitivity could contribute to the pathology of this mutation. Future research on KID syndrome mutations should take care to avoid using a C-terminal tag to track cellular localization and expression or if this is unavoidable, combine this mutation with removal of the 5′ splice site.

Activation of EGFP expression by Cre-mediated excision in a new ROSA26 reporter mouse strain

Blood ◽  
2001 ◽  
Vol 97 (1) ◽  
pp. 324-326 ◽  
Author(s):  
Xiaohong Mao ◽  
Yuko Fujiwara ◽  
Aimée Chapdelaine ◽  
Haidi Yang ◽  
Stuart H. Orkin
Keyword(s):  
Fluorescent Protein ◽  
Threshold Level ◽  
Egfp Expression ◽  
Mouse Strains ◽  
Reporter Strain ◽  
Egfp Gene ◽  
Reporter Mouse ◽  
Functional Studies

Abstract Reporter mouse strains are important tools for monitoring Cre recombinase-mediated excision in vivo. In practice, excision may be incomplete in a given population due to threshold level or variegated expression of Cre. Hence, it is desirable in many experimental contexts to isolate cells that have undergone excision to assess the consequences of gene ablation. To generate alternative reporter mice, an enhanced green fluorescent protein (EGFP) gene was targeted to the retroviral-trapped ROSA26 locus. Upon Cre-mediated excision of “Stop” sequences, EGFP was expressed ubiquitously during embryogenesis and in adult tissues (including T cells, B cells, and myeloid cells). Using this new reporter strain, separation of excised from nonexcised cells in vitro was achieved in thymocytes in a noninvasive manner based on activated EGFP expression. This new EGFP reporter strain should facilitate a variety of conditional gene-targeting experiments, including the functional studies of hematopoietic cells in lineage-specific knockout mice.

scholarly journals Optimization of a Weak 3′ Splice Site Counteracts the Function of a Bovine Papillomavirus Type 1 Exonic Splicing Suppressor In Vitro and In Vivo

2000 ◽  
Vol 74 (13) ◽  
pp. 5902-5910 ◽  
Author(s):  
Zhi-Ming Zheng ◽  
Jesse Quintero ◽  
Eric S. Reid ◽  
Christian Gocke ◽  
Carl C. Baker
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Splicing Factors ◽  
Bovine Papillomavirus ◽  
Splicing Pattern ◽  
In Vitro Splicing

ABSTRACT Alternative splicing is a critical component of the early to late switch in papillomavirus gene expression. In bovine papillomavirus type 1 (BPV-1), a switch in 3′ splice site utilization from an early 3′ splice site at nucleotide (nt) 3225 to a late-specific 3′ splice site at nt 3605 is essential for expression of the major capsid (L1) mRNA. Three viral splicing elements have recently been identified between the two alternative 3′ splice sites and have been shown to play an important role in this regulation. A bipartite element lies approximately 30 nt downstream of the nt 3225 3′ splice site and consists of an exonic splicing enhancer (ESE), SE1, followed immediately by a pyrimidine-rich exonic splicing suppressor (ESS). A second ESE (SE2) is located approximately 125 nt downstream of the ESS. We have previously demonstrated that the ESS inhibits use of the suboptimal nt 3225 3′ splice site in vitro through binding of cellular splicing factors. However, these in vitro studies did not address the role of the ESS in the regulation of alternative splicing. In the present study, we have analyzed the role of the ESS in the alternative splicing of a BPV-1 late pre-mRNA in vivo. Mutation or deletion of just the ESS did not significantly change the normal splicing pattern where the nt 3225 3′ splice site is already used predominantly. However, a pre-mRNA containing mutations in SE2 is spliced predominantly using the nt 3605 3′ splice site. In this context, mutation of the ESS restored preferential use of the nt 3225 3′ splice site, indicating that the ESS also functions as a splicing suppressor in vivo. Moreover, optimization of the suboptimal nt 3225 3′ splice site counteracted the in vivo function of the ESS and led to preferential selection of the nt 3225 3′ splice site even in pre-mRNAs with SE2 mutations. In vitro splicing assays also showed that the ESS is unable to suppress splicing of a pre-mRNA with an optimized nt 3225 3′ splice site. These data confirm that the function of the ESS requires a suboptimal upstream 3′ splice site. A surprising finding of our study is the observation that SE1 can stimulate both the first and the second steps of splicing.

Nobiletin, a polymethoxylated flavonoid from citrus, shows anti-angiogenic activity in a zebrafish in vivo model and HUVEC in vitro model

2011 ◽  
Vol 112 (11) ◽  
pp. 3313-3321 ◽  
Author(s):  
Kai Heng Lam ◽  
Deepa Alex ◽  
In Kei Lam ◽  
Stephen Kwok Wing Tsui ◽  
Zi Feng Yang ◽  
...  
Keyword(s):  
In Vitro Model ◽  
In Vivo Model ◽  
Angiogenic Activity ◽  
Vitro Model

scholarly journals A neuron-specific isoform of brain ankyrin, 440-kD ankyrinB, is targeted to the axons of rat cerebellar neurons.

1995 ◽  
Vol 131 (6) ◽  
pp. 1821-1829 ◽  
Author(s):  
M Kunimoto
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Specific Antibody ◽  
Synthetic Peptide ◽  
Cerebellar Neurons ◽  
Cell Biol ◽  
Insert Region ◽  
Cerebellar Cells

Two isoforms of brain ankyrin, 440- and 220- kD ankyrinB, are generated from the same gene by alternative splicing of pre-mRNA. The larger isoform shares the same NH2-terminal and COOH-terminal domains to the smaller isoform and contains, in addition, a unique inserted domain of about 220-kD in size (Kunimoto, M., E. Otto, and V. Bennett. 1991. J. Cell Biol. 115:1319-1331). Both Isoforms were expressed in primary cerebellar cells in a manner similar to that in vivo; the larger isoform appeared first when axogenesis is actively conducted and the smaller isoform came up later. 440-kD ankyrinB was localized in the axons of cerebellar neurons both in vivo and in vitro using an antibody raised against the insert region, while 220-kD isoform was rather localized in the cell bodies and dendrites of neurons by a specific antibody prepared using a synthetic peptide corresponding to the splice site as antigen. Astroglia cells also expressed 220-kD ankyrinB but not the 440-kD isoform. These results indicate that 440-kD ankyrinB is a neuron-specific isoform targeted to the axons and its unique inserted domain is essential for the targeting.

scholarly journals Control of hnRNP A1 Alternative Splicing: an Intron Element Represses Use of the Common 3′ Splice Site

2000 ◽  
Vol 20 (19) ◽  
pp. 7353-7362 ◽  
Author(s):  
Martin J. Simard ◽  
Benoit Chabot
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Globin Gene ◽  
Single Copy ◽  
Hnrnp A1 ◽  
Internal Exon ◽  
Multiple Copies ◽  
The Common

ABSTRACT Alternative splicing of exon 7B in the hnRNP A1 pre-mRNA produces mRNAs encoding two proteins: hnRNP A1 and the less abundant A1B. We have reported the identification of several intron elements that contribute to exon 7B skipping. In this study, we report the activity of a novel element, conserved element 9 (CE9), located in the intron downstream of exon 7B. We show that multiple copies of CE9 inhibit exon 7B-exon 8 splicing in vitro. When CE9 is inserted between two competing 3′ splice sites, a single copy of CE9 decreases splicing to the distal 3′ splice site. Our in vivo results also support the conclusion that CE9 is a splicing modulator. First, inserting multiple copies of CE9 into an A1 minigene compromises the production of fully spliced products. Second, one copy of CE9 stimulates the inclusion of a short internal exon in a derivative of the human β-globin gene. In this case, in vitro splicing assays suggest that CE9 decreases splicing of intron 1, an event that improves splicing of intron 2 and decreases skipping of the short internal exon. The ability of CE9 to act on heterologous substrates, combined with the results of a competition assay, suggest that the activity of CE9 is mediated by atrans-acting factor. Our results indicate that CE9 represses the use of the common 3′ splice site in the hnRNP A1 alternative splicing unit.

scholarly journals A clinically relevant SCID-hu in vivo model of human multiple myeloma

Blood ◽  
2005 ◽  
Vol 106 (2) ◽  
pp. 713-716 ◽  
Author(s):  
Pierfrancesco Tassone ◽  
Paola Neri ◽  
Daniel R. Carrasco ◽  
Renate Burger ◽  
Victor S. Goldmacher ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Fluorescent Protein ◽  
Severe Combined Immunodeficiency ◽  
Scid Mice ◽  
In Vivo Model ◽  
Fetal Bone ◽  
Experimental Drugs

Abstract We developed a novel in vivo multiple myeloma (MM) model by engrafting the interleukin 6 (IL-6)-dependent human MM cell line INA-6 into severe combined immunodeficiency (SCID) mice previously given implants of a human fetal bone chip (SCID-hu mice). INA-6 cells require either exogenous human IL-6 (huIL-6) or bone marrow stromal cells (BMSCs) to proliferate in vitro. In this model, we monitored the in vivo growth of INA-6 cells stably transduced with a green fluorescent protein (GFP) gene (INA-6GFP+ cells). INA-6 MM cells engrafted in SCID-hu mice but not in SCID mice that had not been given implants of human fetal bone. The level of soluble human IL-6 receptor (shuIL-6R) in murine serum and fluorescence imaging of host animals were sensitive indicators of tumor growth. Dexamethasone as well as experimental drugs, such as Atiprimod and B-B4-DM1, were used to confirm the utility of the model for evaluation of anti-MM agents. We report that this model is highly reproducible and allows for evaluation of investigational drugs targeting IL-6-dependent MM cells in the human bone marrow (huBM) milieu. (Blood. 2005;106:713-716)

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