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Development of in vivo assays to det...

Gestl, Erin Everett.

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Development of in vivo assays to detect mutation in transgenic zebrafish.

Record Type:	Language materials, printed : Monograph/item
Title/Author:	Development of in vivo assays to detect mutation in transgenic zebrafish./
Author:	Gestl, Erin Everett.
Description:	151 p.
Notes:	Adviser: Judith S. Bond.
Contained By:	Dissertation Abstracts International63-05B.
Subject:	Biology, Genetics. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3051653
ISBN:	0493669353

Development of in vivo assays to detect mutation in transgenic zebrafish.
Gestl, Erin Everett.

Development of in vivo assays to detect mutation in transgenic zebrafish. - 151 p.

Adviser: Judith S. Bond.

Thesis (Ph.D.)--The Pennsylvania State University, 2002.

An <italic>in vivo</italic> assay to detect mutations in transgenic zebrafish would aid in the characterization of genomic instability (<italic>gin</italic>) mutants isolated in our lab, and identification of new mutators in zebrafish. To detect mutations in live embryos, green fluorescent protein (GFP) variants were used in a reporter system. The reporter construct we developed for detection of frameshift mutations consisted of a fusion protein containing an in-frame enhanced blue fluorescent protein (EBFP) as an internal control for transgene expression, an SSR susceptible to frameshift mutations, and an out-of-frame enhanced GFP (EGFP) to detect reversion events.

ISBN: 0493669353Subjects--Topical Terms:

1017730
Biology, Genetics.

Development of in vivo assays to detect mutation in transgenic zebrafish.
LDR:03187nam 2200313 a 45 001 934726
005 20110509
008 110509s2002 eng d
020 $a 0493669353
035 $a (UnM)AAI3051653
035 $a AAI3051653
040 $a UnM $c UnM
100 1 $a Gestl, Erin Everett. $3 1258425
245 1 0 $a Development of in vivo assays to detect mutation in transgenic zebrafish.
300 $a 151 p.
500 $a Adviser: Judith S. Bond.
500 $a Source: Dissertation Abstracts International, Volume: 63-05, Section: B, page: 2177.
502 $a Thesis (Ph.D.)--The Pennsylvania State University, 2002.
520 $a An <italic>in vivo</italic> assay to detect mutations in transgenic zebrafish would aid in the characterization of genomic instability (<italic>gin</italic>) mutants isolated in our lab, and identification of new mutators in zebrafish. To detect mutations in live embryos, green fluorescent protein (GFP) variants were used in a reporter system. The reporter construct we developed for detection of frameshift mutations consisted of a fusion protein containing an in-frame enhanced blue fluorescent protein (EBFP) as an internal control for transgene expression, an SSR susceptible to frameshift mutations, and an out-of-frame enhanced GFP (EGFP) to detect reversion events.
520 $a EBFP-EGFP fusion proteins containing mono- and dinucleotide repeats functioned as expected when injected into fertilized eggs. Embryos injected with control (in-frame EGFP) zG4, zG10, and zCA17 constructs fluoresced blue and green, while embryos injected with experimental (out-of-frame EGFP) zG5, zG11, and zCA9 constructs fluoresced blue with rare green fluorescence (<1%).
520 $a Due to the poor fluorescence of EBFP, this gene was replaced with a newly cloned fluorescent protein, DsRed1. When injected, the intensity of red fluorescence was equal to or greater than the green fluorescence of EGFP. DsRed1 constructs, zG10b and zG11b, functioned as anticipated with strong red fluorescence in embryos injected with both constructs and green fluorescence with zG11b. A reversion frequency of 3.5% was achieved following treatment at 25 μg/ml AAF. The different reversion frequency between zG11 and zG11b may be explained by the fact that EBFP and EGFP are nearly identical.
520 $a DsRed1 as a marker for transgene expression functioned well. A new vector, zG17, was constructed and is expected to yield a stronger fluorescent signal. The increase of the mononucleotide tract to 17 should increase the sensitivity of the assay to frameshift mutagens and mutators. The zRec construct also holds the potential of detecting elevated levels of recombination once transgene expression is increased. Transgenic zebrafish containing these constructs could be generated with minimal effort, and serve as independent assays for frameshift mutation and recombination. (Abstract shortened by UMI.)
590 $a School code: 0176.
650 4 $a Biology, Genetics. $3 1017730
650 4 $a Biology, Molecular. $3 1017719
690 $a 0307
690 $a 0369
710 2 0 $a The Pennsylvania State University. $3 699896
773 0 $t Dissertation Abstracts International $g 63-05B.
790 $a 0176
790 1 0 $a Bond, Judith S., $e advisor
791 $a Ph.D.
792 $a 2002
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3051653