The immediate goal of these experiments is to understand the role of agrin in the formation of the embryonic neuromuscular junction. Agrin is a protein made and externalized by the motor nerve terminal that causes the postsynaptic apparatus to form on muscle cells. Currently we are using Xenopus (frog) embryos as an assay system for agrin and other molecules thought to be involved in formation of this synapse. We are assaying the function of agrin isoforms and domains by overexpressing the proteins in the embryo. RNA encoding agrin proteins is injected into early blastomeres of the embryo; it is mixed with RNA encoding the green fluorescent protein (GFP) as a marker. After about 38 hours of development, tadpoles are screened for expression of GFP in the ryot6mal muscles or spinal cord of the trunk. Embryos are fixed and the acetylcholine receptors (AChRs) are stained with rhodamine-bungarotoxin. The AChR aggregates in GFP-positive regions of the embryos are imaged by confocal microscopy. Several Z-series are taken from each group of embryos in an experiment. The volume of AChR aggregates in the myotomes; imaged is quantitated using Metamorph image analysis software. Results to date indicate that overexpression of both neuronal and muscle isoforms of agrin result in a significant increase in AChR aggregate volume. These results suggest that both isoforms of agrin may be important in the formation of the neuromuscular synapse in the developing embryo.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
3P41RR000570-28
Application #
6278511
Study Section
Project Start
1998-07-01
Project End
2000-06-30
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
28
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Malecki, Marek; Putzer, Emily; Sabo, Chelsea et al. (2014) Directed cardiomyogenesis of autologous human induced pluripotent stem cells recruited to infarcted myocardium with bioengineered antibodies. Mol Cell Ther 2:
Malecki, Marek (2014) 'Above all, do no harm': safeguarding pluripotent stem cell therapy against iatrogenic tumorigenesis. Stem Cell Res Ther 5:73
Mavroudi, Maria; Zarogoulidis, Paul; Porpodis, Konstantinos et al. (2014) Stem cells' guided gene therapy of cancer: New frontier in personalized and targeted therapy. J Cancer Res Ther (Manch) 2:22-33
Malecki, Marek; LaVanne, Christine; Alhambra, Dominique et al. (2013) Safeguarding Stem Cell-Based Regenerative Therapy against Iatrogenic Cancerogenesis: Transgenic Expression of DNASE1, DNASE1L3, DNASE2, DFFB Controlled By POLA1 Promoter in Proliferating and Directed Differentiation Resisting Human Autologous Pluripotent J Stem Cell Res Ther Suppl 9:
Malecki, Marek; Tombokan, Xenia; Anderson, Mark et al. (2013) TRA-1-60(+), SSEA-4(+), POU5F1(+), SOX2(+), NANOG(+) Clones of Pluripotent Stem Cells in the Embryonal Carcinomas of the Testes. J Stem Cell Res Ther 3:
Malecki, Marek (2013) Improved targeting and enhanced retention of the human, autologous, fibroblast-derived, induced, pluripotent stem cells to the sarcomeres of the infarcted myocardium with the aid of the bioengineered, heterospecific, tetravalent antibodies. J Stem Cell Res Ther 3:
Malecki, Marek; Dahlke, Jessica; Haig, Melissa et al. (2013) Eradication of Human Ovarian Cancer Cells by Transgenic Expression of Recombinant DNASE1, DNASE1L3, DNASE2, and DFFB Controlled by EGFR Promoter: Novel Strategy for Targeted Therapy of Cancer. J Genet Syndr Gene Ther 4:152
Zarogoulidis, Paul; Darwiche, Kaid; Sakkas, Antonios et al. (2013) Suicide Gene Therapy for Cancer - Current Strategies. J Genet Syndr Gene Ther 4:
Malecki, Marek; Sabo, Chelsea; Putzer, Emily et al. (2013) Recruitment and retention of human autologous CD34+ CD117+ CD133+ bone marrow stem cells to infarcted myocardium followed by directed vasculogenesis: Novel strategy for cardiac regeneration. Mol Cell Ther 1:
Malecki, Marek; Malecki, Bianca (2012) Routing of Biomolecules and Transgenes' Vectors in Nuclei of Oocytes. J Fertili In Vitro 2012:108-118

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