Optimal Conditions for a Transfection Essay

Only available on StudyMode
  • Download(s) : 53
  • Published : May 28, 2013
Open Document
Text Preview
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 transfection essay depends on various conditions. In the experiment the influence of variations in pH of the growth medium is studied as the primary variable. Additionally variation of incubation time during transfection is studied.

Material and methods

Cell line

For the experiment an established Rat-2 cell line in a concentration of 1∙105 cells per dish was cultured in the wells of a six well plate as described in the protocol splitting of cells3.

Transfection

The carrier used was Salmon sperm DNA with a plasmid carrying pEJ (batch 2010) as positive control and with a plasmid carrying 13G61Q wild type N-Ras as negative control. Salmon sperm DNA with a plasmid carrying 61K H-Ras (batch 2010) was used for transfection of the cells to be studied. The transfection medium was HeBS with variable pH values of 6.5, 7.07, and 7.5. Transfection incubation time for pH values 6.5, and 7.5 was four hours. Incubation times for pH value 7.07 were four and 22 hours. After previously mentioned incubation times the transfection medium was replaced by 4% fetal calf serum. Transfection was performed as described in the protocol Transformation3.

Results

Prior to transfection the cells were spread out equally in a homogenous monolayer in all six culture dishes. After addition of the transfection medium the precipitates carrying the plasmids displayed as black dots on top of the cell layer in each culture dish in densities as illustrated in figure 1. Cultures with medium of 6.5 pH containing the plasmid carriers displayed small, low density precipitate particles. The higher the pH value of the medium the larger and higher the density of the precipitates containing it. Accordingly the medium of 7.07 pH displayed medium density precipitate particles. The medium of 7.5 pH displayed large, high density precipitate particles.

| (7.07/22) |(6.5/4) |(7.07/4) | |...
tracking img