Gene cloning involves removing a gene from the genome of an organism and then placing that isolated gene into the genome of a bacterial cell.  The bacterial cell is then responsible for copying and maintaining this foreign gene.  In some cases, the bacterial cell can then transcribe the foreign gene to make a messenger RNA (mRNA) molecule, and the mRNA can then be translated to make a protein product.  Many important human proteins, including insulin (for diabetes patients) and factor VIII (for type A hemophilia patients), have been produced in large quantities by bacterial cells via this gene cloning technique.

Overview

In the early 1970s, scientists isolated the DNA from two different organisms and covalently linked them together in a test tube.  This new hybrid DNA molecule, which contained DNA from two sources, is a recombinant DNA molecule.  This experiment demonstrated the first use of recombinant DNA techniques, methods to manipulate DNA molecules outside of a living organism.  Since then, many advances have made recombinant DNA techniques common practice among biologists. 

Our discussion will focus on a process called gene cloning (figure 12.1). Gene cloning involves isolating a particular gene of interest and inserting that gene into a vector DNA molecule.  Commonly used vectors include the circular plasmid DNA molecules found in bacteria.  The resulting recombinant DNA molecule, composed partly of the gene of interest and partly the plasmid, is then introduced into a host bacterial cell by transformation.  The host bacterial cell maintains and copies this recombinant DNA molecule, so the gene of interest (i.e., the cloned gene) can be studied in more detail.  Once gene cloning is complete, the cloned gene can be used in:

• DNA sequencing experiments. DNA sequencing provides the base pair sequence of the cloned gene.
• Mutagenesis experiments. Mutations can be introduced at desired locations within the cloned gene.  The phenotypic consequences of these mutations can then be studied.
• Protein expression studies. Cloned genes can be expressed via transcription and translation to make a protein product.  Protein expression allows scientists to examine the function of the protein product encoded by a cloned gene or the cloned gene can be expressed at high levels for medical purposes (i.e., to express the human insulin or factor VIII proteins).
• Gene therapy. Cloned genes can be introduced into human cells as a treatment for genetic diseases.

Vectors

As mentioned earlier, gene cloning involves removing a gene of interest from an organism and inserting that gene into a vector DNA molecule (figure 12.2).  The resulting recombinant DNA molecule is then introduced into a host organism for maintenance.  The host can be a bacterium such as Escherichia coli, the yeast Saccharomyces cerevisiae, or in some cases, a virus.

From this point on, let us assume that we are interested in studying the insulin gene isolated from the human genome.  The overall goal of our gene cloning experiment is to insert the human insulin gene into a vector DNA molecule.  In our scenario, the vector will function to:

• Carry the insulin gene (also called an insert or cloned gene).  The insulin gene inserted into a vector can then be recognized and maintained by the host cell.
• Copy the insulin gene. Vectors are capable of efficient DNA replication within the host cell, and host cells often contain many copies of the vector.  Thus, if the insulin gene is inserted in a vector, DNA replication of the vector will produce many copies of the insulin gene in each host cell.

Plasmids often serve as vector molecules. Plasmids (figures 12.1 and 12.2) are small circular DNA molecules found in many bacteria and some eukaryotes.  These plasmids are not part of the chromosome.  Plasmids contain:

• An origin of replication. The origin allows the plasmid to be replicated efficiently within the host cell.  Some origins, such as OriC, allow replication of the plasmid within a particular host cell species (i.e., specifically the bacterium E. coli).  Other origins allow efficient replication in different host species.  The origin also determines the plasmid copy number.  High copy number plasmids contain “strong” origins that allow frequent replication to produce hundreds of plasmid DNA molecules per cell.  Low copy number plasmids have less efficient origins, resulting in relatively few plasmid molecules per cell.
• Unique gene cloning sites. Gene cloning sites are plasmid DNA sequences that function as potential insertion points for the insert DNA sequence (i.e., the human insulin gene).
• Selectable markers. Plasmids contain selectable marker genes that confer an antibiotic resistant phenotype to the host bacterial cell.  Thus, by growing host bacterial cells in the presence of an antibiotic, the researcher can ensure that the bacterial cells contain a plasmid.  Common selectable marker genes include the amp^R gene, which confers resistance to the antibiotic ampicillin, and the tet^R gene, which confers resistance to the antibiotic tetracycline. In the case of the amp^R gene, a bacterial cell that is resistant to ampicillin contains the plasmid; a bacterial cell that is sensitive to ampicillin does not contain the plasmid.  Ampicillin resistant bacteria survive when grown on agar plates that contain ampicillin; ampicillin sensitive bacteria die when grown on agar plates containing ampicillin.
  

Restriction Enzymes

How do we take the insulin gene from a human chromosome and insert it into a vector DNA molecule?

An important tool that is used in the gene cloning process is a restriction enzyme.  Restriction enzymes are endonucleases that recognize specific DNA sequences (restriction enzyme sites) and cleave the phosphodiester bonds within both DNA strands at the restriction enzyme site.  The restriction enzyme site is typically a palindrome DNA sequence.  For example, the restriction enzyme EcoRI isolated from the bacterium E. coli cuts the DNA sequence 5’-GAATTC-3’.  The complementary strand is 3’-CTTAAG-5’, which is identical to the original restriction enzyme sequence but in the reverse orientation (i.e., a palindrome).  EcoRI cuts both DNA strands within this palindromic DNA sequence between the G and A nitrogenous bases. 

The natural function of a restriction enzyme is to protect the bacterial cell from foreign DNA, particularly bacteriophage DNA injected into the bacterial cell during an infection.  Several hundred restriction enzymes have been isolated from bacteria and are available commercially for purchase.

Producing Recombinant DNA Molecules

How can we use the restriction enzyme EcoRI to clone the human insulin gene into a plasmid vector?

Suppose that the restriction enzyme EcoRI recognizes the restriction enzyme sites shown below in both the insulin gene and in the plasmid DNA molecule (see figure 12.3) Note that the plasmid is cut by EcoRI at a single site, while EcoRI cuts on both ends of the human insulin gene (target gene in the figure).

EcoRI cleaves the cloning site within the plasmid and the insulin gene to produce complementary single-stranded regions called sticky ends.  When mixed, the sticky ends of the insulin DNA can form hydrogen bonds with the sticky ends from the vector DNA. 

DNA ligase then forms the final covalent bonds in each DNA strand, covalently linking the insulin gene into the plasmid vector.

A Typical Gene Cloning Experiment

The entire procedure used to clone the insulin gene into a plasmid vector is outlined below (figure 12.4).  For the purposes of this hypothetical experiment, let us assume that the plasmid DNA molecule contains a cloning site, the ampR gene as a selectable marker, OriC, and the lacZ gene (the text below will describe the function of the lacZ gene).

1. The plasmid and chromosome containing the human insulin gene are cut with the same restriction enzyme. A restriction enzyme is chosen to cut the chromosomal DNA into many small pieces.  One of these chromosome pieces contains the insulin gene.  The same restriction enzyme is used to cut the plasmid DNA within the cloning site.  The DNA fragment containing the insulin gene and the plasmid DNA molecule have complementary sticky ends.  
    
2. The cleaved plasmid and the insulin gene are mixed. Three different events can occur:

• • The plasmid sticky ends hydrogen bond with each other. This situation produces an intact plasmid molecule that does not include the insulin insert.
  • Chromosomal DNA fragments that do not include the insulin gene form hydrogen bonds with the plasmid sticky ends. The recombinant DNA molecule produced will contain the wrong insert.
  • The insulin gene fragment forms hydrogen bonds with the plasmid sticky ends.

3. DNA ligase is added. DNA ligase catalyzes the covalent linkage of the insert DNA fragment to the plasmid DNA molecule.  Ligation produces a population of circular DNA molecules, called recombinant DNA molecules, in which an insert is cloned into the plasmid.  Some of these recombinant DNA molecules contain the desired insulin gene insert.

4. Transformation of a host bacterial cell. Now that recombinant DNA molecules have been created, the recombinant DNA molecules are introduced into an E. coli  host cell for maintenance.  When E. coli cells are treated with calcium, the bacteria become competent to take up DNA from the environment.  When the recombinant DNA molecule is added to these competent bacterial cells, and the bacteria are shocked by a brief heat treatment, the recombinant DNA molecule is taken into the cytoplasm of the E. coli (transformation). 
    
5. Host bacteria are grown on an agar plate that contains ampicillin. Two scenarios are possible after transformation: 

• • Some E. coli cells were not transformed (i.e., do not contain a recombinant DNA molecule). These bacterial cells cannot grow on the agar plate containing ampicillin because the bacteria lack the amp^R gene.
  • Some E. coli cells were transformed. Since the plasmid vector contains the amp^R gene, transformed cells are now resistant to ampicillin.  As a result, these transformed cells grow on an agar plate that contains ampicillin.  It is important to note that this population of growing bacteria contains three types of plasmids:
    • Some plasmids lack inserts altogether.
    • Some plasmids contain other chromosomal DNA fragments as the insert (i.e., the wrong insert).
    • Some plasmids contain the insulin gene as the insert (i.e., the correct insert).

6. Identify colonies that contain an insert by the blue-white screening method. Many cloning experiments are designed so that the gene cloning site within the plasmid is located in a DNA sequence called lacZ.  The lacZ gene produces the enzyme β-galactosidase.  Cloning the insert disrupts the lacZ gene, preventing β-galactosidase production.  Disruption of the lacZ gene allows researchers to distinguish bacteria that contain an insert versus bacteria that do not contain an insert.

• • Recombinant plasmid vector without an insert. In this case, the plasmid contains an intact lacZ gene. The plasmid lacZ gene produces β-galactosidase.
  • Recombinant plasmid vector that contains an insert (i.e., the insulin gene). Since the presence of an insert disrupts the lacZ gene, no β-galactosidase is produced.

How do we determine if β-galactosidase is produced?  This is done by plating the bacterial cells on an agar plate that not only includes ampicillin but also includes IPTG, a chemical that activates the lacZ gene to produce the β-galactosidase protein.  The agar plate also contains X-Gal, a chemical substrate for the β-galactosidase enzyme.

• • β-galactosidase is expressed (no insert). The bacterial colony is blue as β-galactosidase converts X-Gal, which is colorless, into a blue product.
  • β-galactosidase is not expressed (insert present). The colony is white because β-galactosidase is nonfunctional and thus cannot convert X-Gal into a blue product.

7. Identify colonies that contain the insulin insert. All the white colonies contain an insert; however, at this point, we cannot distinguish the white colonies that contain the insulin insert from the white colonies that have other DNA fragments as inserts. Several techniques can be used to identify which bacterial cells contain a recombinant vector with an insulin gene.  For example, vectors isolated from white colonies can be cut with the same restriction enzyme used at the beginning of the cloning experiment to release the insert.  The digested DNA can then be analyzed by agarose gel electrophoresis.  The presence of a DNA fragment of the appropriate size for the insulin gene indicates that the chosen colony likely contains the insulin insert.  Alternatively, the polymerase chain reaction (PCR), using primers specific for the insulin gene can be used to amplify the insulin insert.  If a PCR product is produced using these insulin primers, then the insulin gene was cloned successfully.  Finally, determining the nucleotide sequence of the cloned insert by DNA sequencing will determine if the recombinant molecule contains the insulin gene.