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Fate and biological activity of exogenous DNA sequences during serial transfections in NIH/3T3 cells
Abstract:
The efficiency and accuracy of serial transfections in NIH/3T3 fibroblasts were investigated with two plasmids carrying a dominant gene. One plasmid carried the activated ras oncogene of human origin inducing morphological alteration and the oncogenic phenotype of NIH/3T3 cells. The second plasmid carried the bacterial neoR gene conferring resistance to the neomycine analogue G 418. We observed no correlation between the presence of biologically active DNAs in primary transfectants and the capacities of these DNAs to transmit the exogenous information in a second cycle of transfection. Cellular DNA of only two of 13 ras and only 1 of 3 neoR transformants could transform NIH/3T3 in a second cycle of transfections. About half of secondary transfectants, derived from those primary transfectants which did transmit the exogenous DNA, contained apparently complete exogenous sequences and transmitted it efficiently and even with the original site of integration in the host DNA in a third cycle of transfection. Exogenous DNA sequences were amplified in the majority of secondary transfectants but did not enhance biological activity in a third cycle of transfer. The exogenous DNA was found to undergo rearrangements in oncogenic transformants propagated in cell culture.
Insights
Serial transfection efficiency in NIH/3T3 cells varies. While initial gene transfer can be inconsistent, subsequent transfections show stable integration and transmission of exogenous DNA, with some sequences undergoing rearrangements.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Investigating gene transfer efficiency is crucial for understanding genetic manipulation in cell lines.
- NIH/3T3 fibroblasts are a common model for studying oncogene-induced transformation and gene integration.
Purpose of the Study:
- To evaluate the efficiency and accuracy of serial transfections using plasmids encoding the ras oncogene or neomycin resistance (neoR) gene.
- To determine the correlation between biologically active DNA in primary transfectants and their ability to transfer genetic information in subsequent transfection cycles.
Main Methods:
- Serial transfection of NIH/3T3 fibroblasts with two distinct plasmids: one containing a human ras oncogene and another containing the bacterial neoR gene.
- Assessment of transfection efficiency by monitoring morphological alterations (ras) and G 418 resistance (neoR).
- Analysis of exogenous DNA transmission and integration in secondary and tertiary transfection cycles.
Main Results:
- No direct correlation was found between the presence of biologically active DNA in primary transformants and their capacity for secondary transfection.
- Only a small fraction of primary transformants (2/13 ras, 1/3 neoR) could successfully transfer the exogenous DNA in a second cycle.
- Secondary transfectants that efficiently transmitted DNA often contained complete exogenous sequences, integrated stably, and were capable of tertiary transfection.
- Exogenous DNA amplification was observed in most secondary transfectants, but this did not enhance biological activity in tertiary transfer.
- Rearrangements of exogenous DNA sequences occurred in oncogenic transformants during cell culture propagation.
Conclusions:
- Serial transfection efficiency in NIH/3T3 cells is variable, with initial transfer success not always predicting subsequent transferability.
- Stable integration and efficient transmission of exogenous DNA can be achieved in later transfection cycles, particularly when complete sequences are present.
- Exogenous DNA undergoes amplification and rearrangement in cultured cells, which may influence its biological activity and transmission.