12 - Gene Cloning

Gene cloning involves removing a gene from an organism and then placing that isolated gene into the genome of a bacterial cell.  The bacterial cell is then responsible for maintaining this foreign gene.  For example, the bacterial cell copies the foreign gene by DNA replication, transcribes the foreign gene to make messenger RNA (mRNA) molecules, and finally translates the mRNAs to make protein products.  Many medically-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

The gene cloning process was pioneered in 1972, when Paul Berg isolated the DNA from two different organisms and covalently linked them together to form a hybrid DNA molecule in a test tube.  This new hybrid DNA molecule, derived from two sources, is a recombinant DNA molecule.  Berg's 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 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 many bacteria.  The resulting recombinant DNA molecule, composed partly of the gene of interest and partly the plasmid vector, is then introduced into a host bacterial cell by transformation.  The host bacterial cell maintains the 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 structural genes can transcribed and translated to make protein products.  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 make 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 bacterial cell, such as the bacterium Escherichia coli, for maintenance.  

From this point on, let us assume that we are interested in studying the human insulin gene.  The overall goal of our gene cloning experiment is to insert the human insulin gene into a vector DNA molecule and introduce the recombinant DNA molecule via transformation into the bacterium E. coli.  In our scenario, the vector DNA molecule will function to:

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

Plasmids, small circular DNA molecules found in many bacteria, often serve as efficient vector molecules (figures 12.1 and 12.2). In order to make plasmids useful vectors in gene cloning, 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 multiple host species.  The origin also determines the plasmid copy number.  High copy number plasmids contain “strong” origins that allow frequent DNA replication to produce hundreds of plasmid DNA molecules per bacterial cell.  Low copy number plasmids have less efficient origins, resulting in fewer plasmid molecules per bacterial cell.
• Gene cloning sites. Gene cloning sites are plasmid DNA sequences that function as potential insertion sites for the cloned 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 on agar plates in the presence of an antibiotic, the researcher ensures that the bacterial cells contain the 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 the human genome and insert it into a plasmid vector DNA molecule? Restriction enzymes are important molecular tools that assist in the gene cloning process.  Restriction enzymes are endonucleases that recognize and cut specific DNA sequences called restriction enzyme sites.  Restriction enzyme sites are typically palindrome DNA sequences.  For example, the restriction enzyme EcoRI 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 cut foreign DNA that enters a bacterial cell, particularly bacteriophage DNA injected into the bacterial cell during a bacteriophage infection.  Several hundred restriction enzymes have been isolated from bacteria and are available commercially for purchase.

Producing Recombinant DNA Molecules

How can we use an example restriction enzyme, such as EcoRI, to clone the human insulin gene into a plasmid vector DNA molecule? Suppose that EcoRI recognizes restriction enzyme sites on both sides of the insulin gene and at a unique location within the plasmid DNA molecule (see figure 12.3).  

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 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 single restriction enzyme site (cloning site), the ampR gene as a selectable marker, OriC, and the lacZ gene.  The function of the lacZ gene will be described below.

1. The plasmid vector and the chromosome containing the human insulin gene are cut with the same restriction enzyme. A restriction enzyme cuts 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.
  • The insulin gene fragment forms hydrogen bonds with the plasmid sticky ends.  These recombinant DNA molecules contain the correct insert.
  • Chromosomal DNA fragments other than the insulin gene form hydrogen bonds with the plasmid sticky ends. These recombinant DNA molecules produced contain the wrong insert.

3. DNA ligase is added. DNA ligase catalyzes the covalent linkage of the insert DNA fragment to the plasmid DNA molecule.   Some of these recombinant DNA molecules contain the desired insulin gene insert.

4. Transformation of host E. coli cells. The recombinant DNA molecules are now introduced into E. coli  host cells for maintenance via transformation.  During transformation, E. coli cells are treated with calcium, so that 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. 
    
5. Host bacteria are grown on agar plates that contains ampicillin. Two scenarios are possible after transformation: 

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

6. Distinguish bacteria that contain inserts from those who that lack inserts via the blue-white screening method. Many cloning experiments are designed so that the restriction enzyme cutting site within the plasmid is located within a structural gene called lacZ.  The lacZ gene produces the enzyme β-galactosidase.  Cloning the insert into the plasmid vector 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. 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 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 produced (i.e., the plasmid lacks an insert). The bacterial colony is blue as β-galactosidase converts X-Gal, which is colorless, into a blue product.
  • β-galactosidase is not produces (i.e., the plasmid contains an insert). 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 are typically 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.