Cell lines are at the backbone of scientific research and have enabled countless discoveries and preclinical studies Although there are many cell lines available, researchers have many reasons to generate or modify their cell lines—perhaps to study a particular mutation in a cancer cell line or to determine the effects of knocking down a gene of interest.

To generate a stable cell line, there are three general steps:

Three general steps to generate a stable cell line illustration

Though there are different methods of stable cell line generation, each protocol follows these general steps. The most common methods of stable cell line generation include:

  • Transfection
  • Transduction

We will cover the specifics of each of these methods, as well as the advantages and disadvantages of each method in this article. When generating a cell line, it is important to consider if you require transient or stable expression of your gene of interest. Transient expression of a gene occurs when exogenous DNA introduced into a cell line is only temporarily expressed and does not integrate into the genome [1]. Stable expression of a gene means long-term, continued expression of a gene of interest [1]. Transient expression can be a better method when studying short-term effects in cells since this is a much faster method. However, if cells will be used over multiple experiments or studied for long-term changes, then stable expression is preferred [1].

Which cell type should I use?

Before generating a cell line, it is important to decide which type of cell is needed. First, let’s review the types of cells often used in research.

Primary cells

Primary cells are collected directly from patients (such as their blood, tumor, etc.), processed, and cultured. Compared to other cell types, primary cells are more heterogeneous and offer better biological relevance since they are derived directly from patients, though these cells cannot replicate as much and therefore cannot be cultured as long as other cell types [2].

Immortalized cells

Immortalized cells can grow indefinitely in culture [2]. Cells can be immortalized through the introduction of an immortalization gene (such as SV-40 large T antigen, hTERT, etc.), spontaneous immortalization through passaging, or due to an acquired ability to replicate indefinitely as seen in cancer cells [3-6]. The benefit of using an immortalized cell line is that they are much easier to work with in culture and have a much longer ‘lifespan’ as they can be cultured nearly indefinitely. However, immortalized cells often have less biological relevance compare...

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