Optimal Conditions for a Transfection Essay
Optimal conditions for a transfection essay
A study on the effects of variations in transfection conditions on Rat-2 cells
19-2-2010
LUMC
BW3
Marjolein Surstedt (m.surstedt@u-mail.leidenuniv.nl)
0743259
Ewoud Ewing
Abstract
The behavior of a proto-oncogene mutated into an oncogene can be studied in vitro by means of transfection. The success of a transfection essay depends on various conditions. In the experiment the influence of various transfection conditions were studied. An established Rat-2 cell line was transfected with a plasmid containing a mutated Ras gene under various pH conditions. During the focus development stage after successful transfection the cultures developed a fungal infection. Results from previously performed mutual experiments displayed an optimum result with a cell count of 1∙105 cells per culture dish in a transfection medium of 7.07 pH and a four to 22 hours incubation time.
Introduction
The Ras gene is a proto-oncogene found to be mutated in one out of four cancers occurring in humans1. Ras operates as a binary switch in the signaling cascade stimulated by growth factors and therefore performs a key function in switching a cell from a resting state to a state of proliferation2. Mutations in Ras genes generate a hyperactive Ras protein that persists abnormally in its active state thereby transmitting an inappropriate signal for cell proliferation. The behavior of a proto-oncogene mutated into an oncogene can be studied in vitro by means of transfection. In such a transfection essay an oncogene introduced into a suitable tester cell line will drive the transfected cells into cancerous behavior. The tester cell line is selected to be able to thrive in culture and is therefore thought to contain alterations that take them part of the way toward malignancy already1. For this reason introduction of a single oncogene can be the fulfilling step to drive these cells to cancerous behavior. The success of a
A study on the effects of variations in transfection conditions on Rat-2 cells
19-2-2010
LUMC
BW3
Marjolein Surstedt (m.surstedt@u-mail.leidenuniv.nl)
0743259
Ewoud Ewing
Abstract
The behavior of a proto-oncogene mutated into an oncogene can be studied in vitro by means of transfection. The success of a transfection essay depends on various conditions. In the experiment the influence of various transfection conditions were studied. An established Rat-2 cell line was transfected with a plasmid containing a mutated Ras gene under various pH conditions. During the focus development stage after successful transfection the cultures developed a fungal infection. Results from previously performed mutual experiments displayed an optimum result with a cell count of 1∙105 cells per culture dish in a transfection medium of 7.07 pH and a four to 22 hours incubation time.
Introduction
The Ras gene is a proto-oncogene found to be mutated in one out of four cancers occurring in humans1. Ras operates as a binary switch in the signaling cascade stimulated by growth factors and therefore performs a key function in switching a cell from a resting state to a state of proliferation2. Mutations in Ras genes generate a hyperactive Ras protein that persists abnormally in its active state thereby transmitting an inappropriate signal for cell proliferation. The behavior of a proto-oncogene mutated into an oncogene can be studied in vitro by means of transfection. In such a transfection essay an oncogene introduced into a suitable tester cell line will drive the transfected cells into cancerous behavior. The tester cell line is selected to be able to thrive in culture and is therefore thought to contain alterations that take them part of the way toward malignancy already1. For this reason introduction of a single oncogene can be the fulfilling step to drive these cells to cancerous behavior. The success of a
References: 1 Alberts, B. [et. Al](2002) Molecular biology of the cell. Garland Science. New York 2 Weinberg, R.A. (2007) The biology of cancer. Garland Science. New York 3 Protocol for practical PR2 [pic] [pic] [pic] ----------------------- 4