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Dan Weeks, PhD
Professor of Biochemistry and PediatricsCarver College of MedicineUniversity of Iowa51 Newton Rd, 4-710 BSBIowa City, IA 52242
Phone: (319) 335-7918Lab Phone: (319) 335-7919Fax: (319) email@example.com
Figure 1. Whole mount confocal analysis showing innervation (green) of a tadpole ear. Images taken by S.J. Kolker.
The selective inactivation of gene expression is one of the most powerful ways to understand the cellular function of a particular gene product. This approach has been most successfully adopted in mutagenic analysis of organisms ranging from viruses and bacteria to yeast, nematodes, fruit flies and more recently mice. However, as the size and the expense of the organism increases, full mutational analysis becomes less feasible. In fact, for most organisms, it would be difficult to justify the expense and effort required to simulate the genetic approach used in organisms like yeast, fruit flies, zebra fish or mice.
One method to selectively inactivate gene expression in "genetically challenging" organisms is the introduction of oligonucleotides into the cell. There are several ways that oligonucleotides might be used for this purpose. The most commonly used method is to design oligonucleotides that hybridize with specific mRNA molecules leading to degradation of mRNA and loss of de novo protein synthesis.
We are developing and using chemically modified cationic oligonucleotides that enhance the ability of the oligo to act as an antisense agent. We have also shown that these modified oligos can be used to form sequence specific triple helices in vitro under ionic conditions that approximate those found in the nucleus. These promising modifications will be tested for toxicity and efficacy while examining their affect on genes expressed during embryogenesis in the frog Xenopus laevis.
Figure 2. When the transcription factor PitX2c is reduced using cationic antisense oligonucleotide, the developing heart fails to fully execute it's normal asymmetry program. (from Dagle et al. 2003)
We use the cationic oligos and other techniques to examine genes implicated in congenital heart and ear defects. We investigate spatial and temporal expression of genes involved in the development of these two organ systems as well as the early embryonic effects of having mutations that in humans lead to congenital defects. These studies will provide new approaches for the study of gene expression and function in Xenopus laevis, as well as providing a rapid, vertebrate based method for examining gene function.
Bartlett H, Veenstra GJ, Weeks DL. Examining the cardiac NK-2 genes in early heart development. (2010) Pediatr Cardiol. Apr;31(3):335-41.
Li Y, Manaligod JM, Weeks DL (2010). EYA1 mutations associated with the branchio-oto-renal syndrome result in defective otic development in Xenopus laevis. Biol Cell. Feb 17;102(5):277-92.
Allen BG, Weeks DL. (2009) Bacteriophage phiC31 integrase mediated transgenesis in Xenopus laevis for protein expression at endogenous levels. Methods Mol Biol. 518:113-22.
Bartlett HL, Weeks DL. (2008) Lessons from the lily pad: Using Xenopus to understand heart disease. Drug Discov Today Dis Models. Fall;5(3):141-146.
Chesneau, A., Sachs, L.M., Chai, N., Chen, Y., Du Pasquier, L., Loeber, J., Pollet, N., Reilly, M., Weeks, D.L., Bronchain O.J. (2008) Transgenesis procedures in Xenopus. Biol. Cell. Sep 100(9):503-521.
Jacobi, U.G., Akkers, R.C., Pierson, E.S., Weeks, D.L., Dagle, J.M., and Veenstra, G.J. (2007) TBP paralogs accommodate metazoan- and vertebrate-specific developmental gene regulation. EMBO J. Sep 5;26(17):3900-9.
Bartlett, H.L., Sutherland, L., Kolker, S.J., Welp, C., Tajchman, U., Desmarais, V., and Weeks, D.L. (2007) Transient early embryonic expression of Nkx2-5 mutations linked to congenital heart defects in human causes heart defects in Xenopus laevis. Dev Dyn. Sep;236(9):2475-84.
Mitchell, T., Jones, E.A., Weeks, D.L., and Sheets, M.D. (2007) Chordin affects pronephros development in Xenopus embryos by anteriorizing presomitic mesoderm. Develop. Dyn. 236:251-261.
Allen, B.G., Allen-Brady, K., and Weeks, D.L. (2006) Reduction of XNkx2-10 expression leads to anterior defects and malformation of the embryonic heart. Mech. Dev. 123:719-729.
Collop, A.H., Broomfield, J.A.S., Chandraratna, R.A.S., Yong, Z., Deimling, S.J., Kolker, S.J., Weeks, D.L., and Drysdale, T.A. (2006) Retinoic acid signaling is essential for formation of the heart tube in Xenous. Develop. Biol. 291:96-109.
Allen, B.G. and Weeks, D.L. (2006) Using phiC31 integrase to make transgenic Xenopus laevis embryos. Nature Protocols 1:1248-1257.
Knauert, M.P., Lloyd, J.A., Rogers, F.A., Datta, H.J., Bennett, M.L., Weeks, D.L. and Glazer, P.M. (2005) Biochemistry 44:3856-3864.
Bane, B.C., Van Rybroek, J.M., Kolker, S.J., Weeks, D.L., Manaligod, J.M.(2005) EYA1 Expression In The Developing Inner Ear. Annals of Otology, Rhinology and Laryngology.
Kalish, J. M., Seidman, M. M., Weeks, D. L., Glazer, P. M. (2005) Triplex-induced recombination and repair in the pyrimidine motif. Nuc. Acids Res. 33:3492-3502.
Slevin, M.K., Lyons-Levy, G., Weeks, D.L. and Hartley, R.S. (2005) Antisense Knockdown of Cyclin E does not affect the Midblastula Transition in Xenopus laevis Embryos. Cell Cycle 4:1396-1402.
Allen, B.G. and Weeks. D.L. (2005) Transgenic Xenopus laevis C31 integrase.Fembryos can be generated using Nature Methods 2:975-980.
Bartlett, H. L., Scholz, T. D., Lamb, F. S., and Weeks, D. L. (2004). Characterization of embryonic cardiac pacemaker and atrioventricular conduction physiology in Xenopus laevis using non-invasive imaging. Am. J. Physiol, Heart Circ. Physiol. 286: 2035-2041.
Dagle, J.M., Sabel, J.L., Littig, J.L., Sutherland, L.B., Kolker, S.J. and Weeks, D.L. (2003) Pitx2c attenuation results in cardiac defects and abnormalities of intestinal orientation in developing Xenopus laevis. Develop. Biol. 262(2): 268-281.
Hukriede, N.A., Tsang, T.E., Habas, R., Khoo, P.L., Steiner, K., Weeks, D.L., Tam, P.P.L. and Dawid, I.B. (2003) Conserved requirement of Lim1 function for cell movements during gastrulation. Developmental Cell 4: 83-94.
Luo, T., Matsuo-Takasaki,M., Thomas, M.L., Weeks, D.L. and Sargent, T.D. (2002) Transcription Factor AP-2 is an Essential and Direct Regulator of Epidermal Development in Xenopus. Dev. Biol. 245: 136-144.
Dagle, J.M. and Weeks, D.L. (2001) Oligonucleotide Based Strategies to Reduce Gene Expression. Differentiation 69: 75-82.
Vasquez, K.M., Dagle, J.M., Weeks, D.L. and Glazer, P.M (2001) Chromosome targeting at short polypurine sites by cationic triplex-forming oligonucleotides. J. Biol. Chem. 276(42): 38536-38541.