Construction, electroporatic transfer and expression of ZPβypGH and ZPβrtGH in zebrafish

S. G. Sheela; J. D. Chen; S. Mathavan; T. J. Pandian

doi:10.1007/BF02709167

Construction, electroporatic transfer and expression of ZPβypGH and ZPβrtGH in zebrafish

S. G. Sheela, J. D. Chen, S. Mathavan, T. J. Pandian

Neurology

Research output: Contribution to journal › Article › peer-review

8 Scopus citations

Abstract

Recombinant transformation vectors (ZpβypGH and ZpβrtGH) consisting of fish growth hormone cDNA, and a reporter gene β-galactosidase driven by fish promoter (Zp) were constructed. Freshly fertilized eggs of zebrafish (Brachydanio rerio) were electroporated at optimum conditions (0-07 kV voltage; 25 μF capacitance; ∞ ohm resistance and 2 pulses) in the presence of one of these transformation vectors (100 μg circular DNA/ml). In either cases 72% of the electroporated eggs successfully hatched, in comparison to the 85% hatchability of the control eggs. Genomic DNA extracted from fins of randomly chosen F₀ individuals was screened (by Southern blot hybridization); the transgenes were retained in the host genome of all the randomly chosen adult transformants. Fin-positive presumptive founder parents were crossed with control counterparts and the DNA of randomly chosen F₁ progenies was screened for germ-line transformation. Southern analysis of chosen F₁ progenies revealed the persistence of ZpβypGH or ZpβrtGH in 53% of the F₁ progenies. Southern analyses of chosen F₁ progenies and the frequency (53% of F₁ ZpβrtGH and 53% of F₁ ZpβypGH) of transmission revealed the degree of mosaicism in F₀ transformants. Expression was confirmed from the 3-4 times elevated levels of activity of the reporter gene and 30-40% accelerated growth of transgenic F₀ and F₁ progenies.

Original language	English (US)
Pages (from-to)	565-576
Number of pages	12
Journal	Journal of Biosciences
Volume	23
Issue number	5
DOIs	https://doi.org/10.1007/BF02709167
State	Published - 1998

Keywords

All-fish genes
Electroporation
Reporter gene expression
Transgenesis
Transmission
Zebrafish

ASJC Scopus subject areas

Biochemistry, Genetics and Molecular Biology(all)
Agricultural and Biological Sciences(all)

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10.1007/BF02709167

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@article{b77faf43e8ca47ff9749378d794ec5e6,

title = "Construction, electroporatic transfer and expression of ZPβypGH and ZPβrtGH in zebrafish",

abstract = "Recombinant transformation vectors (ZpβypGH and ZpβrtGH) consisting of fish growth hormone cDNA, and a reporter gene β-galactosidase driven by fish promoter (Zp) were constructed. Freshly fertilized eggs of zebrafish (Brachydanio rerio) were electroporated at optimum conditions (0-07 kV voltage; 25 μF capacitance; ∞ ohm resistance and 2 pulses) in the presence of one of these transformation vectors (100 μg circular DNA/ml). In either cases 72% of the electroporated eggs successfully hatched, in comparison to the 85% hatchability of the control eggs. Genomic DNA extracted from fins of randomly chosen F0 individuals was screened (by Southern blot hybridization); the transgenes were retained in the host genome of all the randomly chosen adult transformants. Fin-positive presumptive founder parents were crossed with control counterparts and the DNA of randomly chosen F1 progenies was screened for germ-line transformation. Southern analysis of chosen F1 progenies revealed the persistence of ZpβypGH or ZpβrtGH in 53% of the F1 progenies. Southern analyses of chosen F1 progenies and the frequency (53% of F1 ZpβrtGH and 53% of F1 ZpβypGH) of transmission revealed the degree of mosaicism in F0 transformants. Expression was confirmed from the 3-4 times elevated levels of activity of the reporter gene and 30-40% accelerated growth of transgenic F0 and F1 progenies.",

keywords = "All-fish genes, Electroporation, Reporter gene expression, Transgenesis, Transmission, Zebrafish",

author = "Sheela, {S. G.} and Chen, {J. D.} and S. Mathavan and Pandian, {T. J.}",

note = "Funding Information: SGS records her grateful appreciation to the Asian Fisheries Society, Manila, for a travel grant which enabled her researcha t Keelung, Taiwan. Financial support extended by the Indian Council for Agricultural Research, Council of Science and Industrial Research and Departmento f Biotechnology, New Delhi is gratefully acknowledged.",

year = "1998",

doi = "10.1007/BF02709167",

language = "English (US)",

volume = "23",

pages = "565--576",

journal = "Journal of Biosciences",

issn = "0250-5991",

publisher = "Springer India",

number = "5",

}

TY - JOUR

T1 - Construction, electroporatic transfer and expression of ZPβypGH and ZPβrtGH in zebrafish

AU - Sheela, S. G.

AU - Chen, J. D.

AU - Mathavan, S.

AU - Pandian, T. J.

N1 - Funding Information: SGS records her grateful appreciation to the Asian Fisheries Society, Manila, for a travel grant which enabled her researcha t Keelung, Taiwan. Financial support extended by the Indian Council for Agricultural Research, Council of Science and Industrial Research and Departmento f Biotechnology, New Delhi is gratefully acknowledged.

PY - 1998

Y1 - 1998

N2 - Recombinant transformation vectors (ZpβypGH and ZpβrtGH) consisting of fish growth hormone cDNA, and a reporter gene β-galactosidase driven by fish promoter (Zp) were constructed. Freshly fertilized eggs of zebrafish (Brachydanio rerio) were electroporated at optimum conditions (0-07 kV voltage; 25 μF capacitance; ∞ ohm resistance and 2 pulses) in the presence of one of these transformation vectors (100 μg circular DNA/ml). In either cases 72% of the electroporated eggs successfully hatched, in comparison to the 85% hatchability of the control eggs. Genomic DNA extracted from fins of randomly chosen F0 individuals was screened (by Southern blot hybridization); the transgenes were retained in the host genome of all the randomly chosen adult transformants. Fin-positive presumptive founder parents were crossed with control counterparts and the DNA of randomly chosen F1 progenies was screened for germ-line transformation. Southern analysis of chosen F1 progenies revealed the persistence of ZpβypGH or ZpβrtGH in 53% of the F1 progenies. Southern analyses of chosen F1 progenies and the frequency (53% of F1 ZpβrtGH and 53% of F1 ZpβypGH) of transmission revealed the degree of mosaicism in F0 transformants. Expression was confirmed from the 3-4 times elevated levels of activity of the reporter gene and 30-40% accelerated growth of transgenic F0 and F1 progenies.

AB - Recombinant transformation vectors (ZpβypGH and ZpβrtGH) consisting of fish growth hormone cDNA, and a reporter gene β-galactosidase driven by fish promoter (Zp) were constructed. Freshly fertilized eggs of zebrafish (Brachydanio rerio) were electroporated at optimum conditions (0-07 kV voltage; 25 μF capacitance; ∞ ohm resistance and 2 pulses) in the presence of one of these transformation vectors (100 μg circular DNA/ml). In either cases 72% of the electroporated eggs successfully hatched, in comparison to the 85% hatchability of the control eggs. Genomic DNA extracted from fins of randomly chosen F0 individuals was screened (by Southern blot hybridization); the transgenes were retained in the host genome of all the randomly chosen adult transformants. Fin-positive presumptive founder parents were crossed with control counterparts and the DNA of randomly chosen F1 progenies was screened for germ-line transformation. Southern analysis of chosen F1 progenies revealed the persistence of ZpβypGH or ZpβrtGH in 53% of the F1 progenies. Southern analyses of chosen F1 progenies and the frequency (53% of F1 ZpβrtGH and 53% of F1 ZpβypGH) of transmission revealed the degree of mosaicism in F0 transformants. Expression was confirmed from the 3-4 times elevated levels of activity of the reporter gene and 30-40% accelerated growth of transgenic F0 and F1 progenies.

KW - All-fish genes

KW - Electroporation

KW - Reporter gene expression

KW - Transgenesis

KW - Transmission

KW - Zebrafish

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U2 - 10.1007/BF02709167

DO - 10.1007/BF02709167

M3 - Article

AN - SCOPUS:0032430722

SN - 0250-5991

VL - 23

SP - 565

EP - 576

JO - Journal of Biosciences

JF - Journal of Biosciences

IS - 5

ER -

Construction, electroporatic transfer and expression of ZPβypGH and ZPβrtGH in zebrafish

Abstract

Keywords

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