Chapter 7 – Complementary Experimental Tools  275

in the cell membrane to pump DNA from the outside to the inside). This natural phenom­

enon in bacteria occurs in a process called “horizontal gene transfer,” which results in genetic

diversity through the transfer of plasmid DNA between different cells, and is, for example, a

mechanism for propagating antibiotic resistance in a cell population. It may also have evolved

as a mechanism to assist in the repair of damaged DNA, that is, to enable the internaliza­

tion of nondamaged DNA that can then be used as a template from which to repair native

damaged DNA.

Artificial methods can improve the rate of transformation. These can include treating cells

first with enzymes to strip away outer cells walls, adding divalent metal ions such as mag­

nesium or calcium to increase binding of DNA (which has a net negative charge in solution

due to the presence of the backbone of negatively charged phosphate groups), or increasing

cell membrane fluidity. These also include methods that involve a combination of cold and

heat shocking cells to increase internalization of recombinant by undetermined mechanisms

as well as using ultrasound (sonication) to increase the collision frequency of recombinant

DNA with host cells. The most effective method, however, is electroporation. This involves

placing the aqueous suspension of host cells and recombinant DNA into an electrostatic

field of strength 10–​20 kV cm⁻¹ for a few milliseconds that increases the cell membrane

permeability dramatically through creating transient holes in the membrane through which

plasmid DNA may enter.

Transfection can be accomplished using an extensive range of techniques, some of which

are similar to those used for transformation, for example, the use of electroporation. Other

more involved methods have been optimized specifically for host animal cell transfection,

however. These include biochemical-​based methods such as packaging recombinant DNA

into modified liposomes that then empty their contents into a cell upon impact on, and mer­

ging with, the cell membrane. A related method is protoplast fusion, which involves chem­

ically or enzymatically stripping away the cell wall from a bacterial cell to enable it to fuse in

suspension with a host animal cell. This delivers the vector that may be inside the bacterial

cell, but with the disadvantage of delivery of the entire bacterial cell contents, which may

potentially be detrimental to the host cell.

But there are also several biophysical techniques for transfection. These include

sonoporation (using ultrasound to generate transient pores in cell membranes), cell squeezing

(gently massaging cells through narrow flow channels to increase the membrane perme­

ability), impalefection (introducing DNA bound to a surface of a nanofiber by stabbing the

cell), gene guns (similar to impalefection but using DNA bound to nanoparticles that are fired

into the host cell), and magnet-​assisted transfection or magnetofection (similar to the gene

gun approach, though here DNA is bound to a magnetic nanoparticle with an external B-​field

used to force the particles into the host cells).

The biophysical transfection tool with the most finesse involves optical transfection, also

known as photoporation. Here, a laser beam is controllably focused onto the cell membrane

generating localized heating sufficient to form a pore in the cell membrane and allow recom­

binant DNA outside the cell to enter by diffusion. Single-​photon absorption processes in

the lipid bilayer can be used here, centered on short wavelength visible light lasers; how­

ever, better spatial precision is enabled by using a high-​power near-​infrared (IR) femtosecond

pulsed laser that relies on two-​photon absorption in the cell membrane, resulting in smaller

pores and less potential cell damage.

Viruses undergoing transfection (i.e., viral transduction) are valuable because they can

transfer genes into a wide variety of human cells in particular with very high transfer rates.

However, this method can also be used for other cell types, including bacteria. Here, the

recombinant DNA is packaged into an empty virus capsid protein coat (see Chapter 2). The

virus then performs its normal roles of attaching to host cell and then injecting the DNA into

the cell very efficiently, compared to the other transfection/​transformation methods.

The process of inserting recombinant DNA into a host cell has normally low efficiency,

with only a small proportion of host cells successfully taking up the external DNA. This

presents a technical challenge in knowing which cells have done so, since these are the ones

that need to be selectively cultivated from a population. This selection is achieved by engin­

eering one or more selectable markers into the vector. A selectable marker is usually a gene