The molecular cloning of a hyperactive piggybac transposase and its transfection into hek293t cells for the purpose of protein purification
The molecular cloning of a hyperactive piggybac transposase and its transfection into hek293t cells for the purpose of protein purification
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2013-08
Authors
Macias, Jessie Lin
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[Honolulu] : [University of Hawaii at Manoa], [August 2013]
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Abstract
Traditionally, a virus with genes of interest is generated to integrate its cargo into the host genome. More recently however, has been the advancement of harnessing the ability of naturally occurring transposases and constructed plasmids to cut and paste genes of interest into the host genome, also creating genetically modified organisms. A transposase is a protein that has specific recognition sequences that it binds to; cuts out all DNA (to be the transposon) in between these two flanking recognition sequences and then inserts it into the host chromosomal DNA. The goal of this work was to create a new method of gene delivery in embryos, by injecting purified hyperactive piggyBac transposase (HyPBase) into embryos, instead of the DNA that codes for it, thereby excluding the time needed for transcription and translation to occur.
We first created a HyPBase vector with a histidine tag to aid in the transposase protein detection by antibody. Then we produced a stable HyPBase expression cell line by transfecting human embryonic kidney (HEK) cells with our cloned vector. The resulting protein was then purified using streptavidin resins according to a tandem affinity purification (TAP) protocol. The purified HyPBase protein was then injected into mice embryos by pronuclear microinjection, cytoplasmic microinjection and intracytoplasmic sperm injection methods. The 2-cell stage embryos were transferred to surrogate mothers and 2 of 17 live pups were transgenic. This will result in more rapid gene delivery, and lower the percentage of mosaicism in transgenic animals.
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M.S. University of Hawaii at Manoa 2013.
Includes bibliographical references.
Includes bibliographical references.
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microinjection
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Theses for the degree of Master of Science (University of Hawaii at Manoa). Developmental and Reproductive Biology.
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