| Summary: | 碩士 === 國立海洋大學 === 水產養殖學系 === 84 === ABSTRACT Gene targeting is a
technique for homologous recombination through the gene
introduction, and can be applied on the recombination of gene
knock-out and gene therapy. Previous studies on gene targeting
were usually focused on murine embryonic stem cells to
cultivate chimeric mice, which is helpful forfunction analysis
of mouse genes. However, since up to now, no studies of gene
targeting on fish was reported yet, the objective of this
research is to investigate the gene targeting on fish. We
designed recombinant vectors using zebrafish insulin-like
growth factor I(IGF-I) as target gene and designed recombinant
vectors working on zebrafish liver cells. Ultimately, we
expected to achieve the goal of gene targeting, to improve the
successful rate of gene transfer, and to study the functions of
IGF-I in liver cells. IGF-I is found in most organisms. It
is a member of insulin gene family and with functions of cell
division, differentiation, and growth. It also playsan important
role on regulations of metabolism and osmotic pressure. Since
stu-dies of IGF-I 0n fish are still not as clear as on mammals,
we try to figure out the gene structure and functions of IGF of
fish. Colleague of our laboratory hascloned two IGF-I gene with
similar nucleic acid sequence from zebrafish genome.The vector
for gene targeting included exon I and exon II as well as intron
I and II of IGF-I gene, and the reporter gene we used for
positive selection was Neo geng with PGK promoter, and TK gene
of SHV for negative selection. Further- more, the Neo gene was
designed to ligate with exon I in order to block the ex-pression
of IGF-I gene. In addition, to realize the effects of the
sequence length and promoter region on the recombination
efficiency, we designed two vectors; one included the 650 bp
5'untranslated region only, and the order con- tained the 1400
bp promoter region as well as the 5'untranslated region. The
targeting vector, from twice CsCl purification, was linearized
by restriction enzyme Sal I, and transfected into zebrafish
liver cells by electroporation or calcium phosphate
precipitation for selection of gene targeted stable clones.
After G418 and gancyclovir, the stable clones were analyzed by
polymerase chain reaction or Southern hybridization.
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