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https://hdl.handle.net/2142/83529
Description
Title
Liposome-Mediated DNA Transfer
Author(s)
Russell, Reb John, II
Issue Date
1997
Doctoral Committee Chair(s)
Wheeler, Matthew B.
Department of Study
Animal Sciences
Discipline
Animal Sciences
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Language
eng
Abstract
The production of transgenic animals carrying a single transgene is very expensive. Two novel methods to deliver transgenes to animals were examined. The first method involved the use of high pressure to increase transfection efficiency in cell lines as a potential way to produce transgenics through embryonic stem cells. The second method used artificial insemination with liposome-treated sperm in an effort to produce transgenic animals. The effects of high pressure on cell viability was examined in three cell lines in triplicate using forskolin stimulated cAMP production and trypan blue exclusion. Chinese hamster ovary (CHO), porcine kidney (PK1) and mouse embryonic stem cell (ESD3) cell lines were placed under various pressures in a hydraulic pressure bomb chamber. The effective dose that killed half of the cells (ED50) for viability of the CHO, PK1 and ESD3 cell lines were 25,607 +/- 1005, 26,869 +/- 4252, and 15,436 +/- 210 pounds per square inch (psi), respectively. Based on the results, beta-Galactosidase activity was assayed as a measure of transfection efficiencies at 10,000 psi compared with atmospheric pressure. Three days following liposome transfections cells were assayed beta-Galactosidase activity and transfection efficiencies increased (p < 0.05) at 10,000 psi and 44°C as compared with 37°C at atmospheric pressures in CHO cells. The initial artificial insemination experiments were designed to determine if the liposome complex interfered with fertilization. Swine oocytes were incubated with liposome/DNA treated sperm and were examined for male pronuclear development. The results for three experiments indicate that 93.9% of the oocytes underwent maturation (81.7% for controls), 84.8% of the matured oocytes were penetrated (90.0% for controls) and 84.6% of the penetrated oocytes had a male pronucleus (100% for controls). The second part, females swine were inseminated with sperm treated with liposome/DNA complexes. Blastocysts were recovered from the uterus eight days after artificial insemination, lysed and subjected to PCR analysis for the neo gene. Ninety-five percent of the embryos recovered contained the neo sequence with controls being PCR negative. These results showed that both the use of high pressure for cell transfection and artificial insemination of oocytes with liposome-treated sperm were effective methods for the delivery of DNA. These methods may provide new means to produce transgenic animals.
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