Parking . . .
>>Published online before print September 23, 2002
Proc. Natl. Acad. Sci. USA, 10.1073/pnas.202471899
Plant Biology
Regulation of transgene expression in plants with polydactyl zinc finger transcription factors
M. Isabel Ordiz *, Carlos F. Barbas III , and Roger N. Beachy *
*Donald Danforth Plant Science Center, 975 North Warson Road, St. Louis, MO 63132; and The Skaggs Institute for Chemical Biology and Department of Molecular Biology, The Scripps Research Institute, BCC-550, North Torrey Pines Road, La Jolla, CA 92037
Contributed by Roger N. Beachy, August 6, 2002
Designer zinc finger transcription factors (TFsZF) have been developed to control the expression of transgenes and endogenous genes in mammalian cells. Application of TFsZF technology in plants would enable a wide range of both basic and applied studies. In this paper, we report the use of TFsZF to target a defined 18-bp DNA sequence to control gene expression in plant cells and in transgenic plants. A ß-glucuronidase reporter gene was activated by using the designed six-zinc finger protein 2C7 expressed as a fusion with the herpes simplex virus VP16 transcription factor activation domain. Reporter gene expression was activated 5- to 30-fold by using TFsZF in BY-2 protoplasts, whereas expression was increased as much as 450 times in transgenic tobacco plants. Use of a phloem-specific promoter to drive expression of the TFsZF resulted in activation of the reporter gene in vascular tissues. Transgenic tobacco plants that produce 2C7 transcription factors were phenotypically normal through two generations, suggesting that the factors exerted no adverse effects. This study demonstrates the utility of zinc finger technology in plants, setting the stage for its application in basic and applied agricultural biotechnology.<<
>>Published online before print September 23, 2002
Proc. Natl. Acad. Sci. USA, 10.1073/pnas.192412899
Plant Biology
Heritable endogenous gene regulation in plants with designed polydactyl zinc finger transcription factors
Xuen Guan *, Justin Stege ¶, Myoung Kim *, Zina Dahmani *, Nancy Fan *||, Peter Heifetz *, Carlos F. Barbas III , and Steven P. Briggs *
*Torrey Mesa Research Institute, San Diego, CA 92121; and The Skaggs Institute for Chemical Biology and Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037
Contributed by Steven P. Briggs, July 10, 2002
Zinc finger transcription factors (TFsZF) were designed and applied to transgene and endogenous gene regulation in stably transformed plants. The target of the TFsZF is the Arabidopsis gene APETALA3 (AP3), which encodes a transcription factor that determines floral organ identity. A zinc finger protein (ZFP) was designed to specifically bind to a region upstream of AP3. AP3 transcription was induced by transformation of leaf protoplasts with a transformation vector that expressed a TFZF consisting of the ZFP fused to the tetrameric repeat of herpes simplex VP16's minimal activation domain. Histochemical staining of ß-glucuronidase (GUS) activity in transgenic AP3::GUS reporter plants expressing GUS under control of the AP3 promoter was increased dramatically in petals when the AP3-specific TFZF activator was cointroduced. TFZF-amplified GUS expression signals were also evident in sepal tissues of these double-transgenic plants. Floral phenotype changes indicative of endogenous AP3 factor coactivation were also observed. The same AP3-specific ZFPAP3 was also fused to a human transcriptional repression domain, the mSIN3 interaction domain, and introduced into either AP3::GUS-expressing plants or wild-type Arabidopsis plants. Dramatic repression of endogenous AP3 expression in floral tissue resulted when a constitutive promoter was used to drive the expression of this TFZF. These plants were also sterile. When a floral tissue-specific promoter from APETALA1 (AP1) gene was used, floral phenotype changes were also observed, but in contrast the plants were fertile. Our results demonstrate that artificial transcriptional factors based on synthetic zinc finger proteins are capable of stable and specific regulation of endogenous genes through multiple generations in multicellular organisms.<<
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