图文详细电转化原理

Molecular Tool: Electroporation

Electroporation

is

a

mechanical

method

used

to

introduce

polar

molecules

into a host cell through the cell membrane. In this procedure, a large

electric pulse temporarily disturbs the phospholipid bilayer, allowing

molecules like DNA to pass into the cell (Purves et. al., 2001).

Background

Many research techniques in molecular biology require a foreign gene or

protein

material

to

be

inserted

into

a

host

cell.

Since

the

phospholipid

bilayer of the plasma membrane has a hydrophobic exterior and a

hydrophobic interior (Fig. 1), any polar molecules, including DNA and

protein,

are

unable

to

freely

pass

through

the

membrane

(Farabee,

2001).

Figure 1. Diagram of the Phospholipid Bilayer.

This image shows the chemical

components of the plasma membrane. The polar head groups face outward while

the hydrophobic tail groups face inward and interact with one another to hold the

membrane together. Polar molecules cannot pass through this membrane without

external aid (Farabee, 2001). Image from

/faculty/farabee/BIOBK/

Permission Pending.

Many methods have been developed to surpass this barrier and allow the

insertion of DNA and other molecules into the cells to be studied. One

such method is electroporation.

The

concept

of

electroporation

capitalizes

on

the

relatively

weak

nature

of the phospholipid bilayer's hydrophobic/hydrophilic interactions and

its ability to spontaneously reassemble after disturbance (Purves,

et.

al.,

2001).

Thus,

a

quick

voltage

shock

may

disrupt

areas

of

the

membrane

temporarily,

allowing

polar

molecules

to

pass,

but

then

the

membrane

may

reseal quickly and leave the cell intact.

Procedure

The host cells and the molecules to be inserted into these cells are

suspended in solution. The electroporation apparatus is typically

commercially produced and purchased, but the basic process inside such

an apparatus may be represented in a schematic diagram (Fig 2).

Figure 2. Diagram of the basic circuit setup of the electroporation

apparatus.

This diagram shows the basic electric circuit that provides the voltage

for electroporation. Diagram idea from

/~melcher/MG/MGW4/

When the first switch is closed, the capacitor charges up and stores a

high voltage. When the second switch is closed, this voltage discharges

through the liquid of the cell suspension (Melcher, 2000). Typically,

10,000-100,000 V/cm (varying with cell size) in a pulse lasting a few

microseconds to a millisecond is necessary for electroporation. This

electric pulse disturbs the phospholipid bilayer of the membrane and

causes the formation of temporary aqueous pores. The electric potential

across the membrane of the cell simultaneously rises by about 0.5-1.0 V

so that charged molecules (such as DNA) are driven across the membrane

through the pores in a manner similar to electrophoresis (Fig 3).

Figure 3. Graphic representation of plasmids containing a foreign DNA

insert passing through temporary aqueous pores in the plasma membrane.

The actual entry of DNA into the cell cannot be observed with a microscope, but this

artist's rendering shows the basic concept of the formation of pores in the

membrane through which DNA can pass (Maxcyte, 2002). Image from

/

Permission pending.

As charged ions and molecules flow through the pores, the cell membrane

discharges and the pores quickly close, and the phospholipid bilayer

reassembles (Weaver, 1995). The intended molecules should now be inside

the cell for further use or study.

Advantages and Disadvantages of Electroporation

Several methods other than electroporation are used to transfer polar

molecules like DNA into host cells. These other methods include

microprecipitates, microinjection, liposomes, and biological vectors.

(Melcher, 2000). Electroporation has both advantages and disadvantages

compared to these methods.

Advantages:

?

Versatility

: Electroporation is effective with nearly all cell and

species types (Nickoloff, 1995).

?

Efficiency:

A

large

majority

of

cells

take

in

the

target

DNA

or

molecule.

In a study on electrotransformation of E. coli, for example, 80% of the

cells received the foreign DNA (Miller and Nickoloff, 1995).

?

Small Scale:

The amount of DNA required is smaller than for other

methods (Withers, 1995)

?

In vivo

:

The procedure may be performed with intact tissue (Weaver,

1995). A paper published in

Developmental Biology

showed the successful

transfer

of

a

DNA

construct

with

a

fluorescent

reporter

gene

into

intact

mouse brain tissue (Fig 4) (Saito, 2001).

Figure 4. Image of

in vivo

electroporation in a mouse brain.

The mouse

brains (telencephalons) in these images are expressing reporter genes (EYFP)

introduced in gene constructs by electroporation. Image from

/rc01/in_vivo_

Permission

pending.

Disadvantages:

?

Cell Damage:

If

the pulses are

of the wrong

length or intensity,

some

pores may become too large or fail to close after membrane discharge

causing cell damage or rupture (Weaver, 1995).

?

Nonspecific Transport:

The transport of material into and out of the

cell during the time of electropermeability is relatively nonspecific.

This

may

result

in

an

ion

imbalance

that

could

later

lead

to

improper

cell

function and cell death (Weaver, 1995)

Applications

As

previously

mentioned,

electroporation

is

widely

used

in

many

areas

of

molecular biology research and in the medical field. Some applications

of electroporation include:

-

DNA

Transfection

or

Transformation

:

This

is

likely

the

most

widespread

use of electroporation. Specific genes can be cloned into a plasmid and

then

this

plasmid

introduced

into

host

cells

(bacterial

or

otherwise)

in

order

to

investigate

gene

and

protein

structure

and

function.

(Nickoloff,

1995)

Figure 5. Microscope images of the results of transfection by

electroporation.

In this experiment, a gene construct was inserted by

electroporation into the cells shown on the right. The fluorescence of the protein

produced by the reporter gene included in this construct shows that the DNA was

properly uptaken in the majority of cells. These cells could now be used in further

experimentation (MaxCyte, 2002). Image from

/

Permission pending.