12 - Gene Cloning

Gene cloning involves removing a gene from the genome of ay organism and then placing that gene into a 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 successfully by bacterial cells via this gene cloning technique.

Gene Cloning (overview)

In the early 1970s, scientists isolated the DNA from two different organisms and covalently linked them together in a test tube.  This new 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 another DNA molecule, called a vector.  A common vector is a small circular plasmid DNA molecule isolated from bacteria (see below).  This recombinant DNA molecule, composed partly of the gene of interest and partly the microbe DNA molecule, is then introduced into a host cell (typically a bacterial cell).  The host cell maintains and copies this recombinant DNA molecule, so the gene of interest (i.e., a 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 in bacteria).
• Gene therapy. Cloned genes can be introduced into human cells as a treatment for diseases caused by mutations.

Vectors

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 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 β-globin gene isolated from the human genome.  The overall goal of our gene cloning experiment is to insert the human β-globin gene into a vector DNA molecule.  In our scenario, the vector will function to:

• Carry the β-globin gene (also called an insert) within a host cell. When the β-globin gene is inserted into a vector, it can be recognized and maintained by the host cell.
• Copy the β-globin 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 β-globin gene is inserted in a vector, many copies of the β-globin gene will be present in each host cell.

What types of molecules can function as vectors?

• Plasmids. 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 bacterial 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 100 – 200 plasmid molecules per cell.  Low copy number plasmids have less efficient origins, resulting in 1 – 2 plasmid molecules per cell.
  • Unique gene cloning sites. Gene cloning sites are DNA sequences that function as the insertion point for the insert into the plasmid.
  • Selectable markers. Plasmids contain selectable marker genes that confer an antibiotic resistant phenotype to the host cell.  Thus, by growing host cells in the presence of an antibiotic, the researcher can ensure that each host cell contains 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 host cell that is resistant to ampicillin contains the plasmid; a host cell that is sensitive to ampicillin does not contain the plasmid.  Ampicillin resistant cells survive on agar plates that contain ampicillin; ampicillin sensitive cells die on agar plates containing ampicillin.
• Viral DNA. The gene of interest (β-globin) can be inserted into the genetic material of a virus.  When the virus infects a host cell, the viral genetic material along with the β-globin gene will be copied by the host cell.

Restriction Enzymes

How do we take the β-globin gene from the chromosome of a human cell 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 a specific DNA sequence (a restriction enzyme site) and cleaves the phosphodiester bonds within both strands of the DNA molecule at the recognition enzyme site.  The restriction enzyme site is typically a palindrome DNA sequence.  As an example, suppose that 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 site. 

Restriction enzymes are made by many bacteria to protect the cell from foreign DNA, particularly bacteriophage DNA injected into a bacterium during an infection.  Several hundred restriction enzymes have been isolated from bacteria and are available for purchase.

Producing Recombinant DNA Molecules

How can we use the restriction enzyme EcoRI to clone the β-globin gene into a plasmid vector?

Suppose that the restriction enzyme EcoRI recognizes the restriction enzyme site shown below in both the β-globin gene and in the plasmid DNA molecule (see figure 12.3).

EcoRI cleaves the cloning site within the plasmid and the β-globin molecules to produce complementary single-stranded regions called sticky ends.  Thus, when mixed, the sticky ends of the β-globin 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, linking the insert (β-globin) to the vector (plasmid).

A Gene Cloning Experiment

Recall that in a gene cloning experiment, the goal is to insert a gene of interest (i.e., the β-globin gene) into a vector DNA molecule.  The approach used to clone the β-globin gene is outlined below and is shown in figure 12.4:

1. The plasmid and chromosomal DNA molecule containing the β-globin gene are digested 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 β-globin gene.  The same restriction enzyme is used to cut the plasmid DNA within the cloning site.  The DNA fragment containing the β-globin gene and the plasmid DNA molecule have complementary sticky ends.  For the purposes of this hypothetical experiment, let us assume that the plasmid DNA molecule contains the amp^R gene as a selectable marker.
    
2. The cleaved plasmid and β-globin gene fragment 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 β-globin insert.
  • Chromosomal DNA fragments that do not include the β-globin gene form hydrogen bonds with the plasmid sticky ends. The recombinant DNA molecule produced will contain the wrong insert.
  • The β-globin gene fragment forms hydrogen bonds with the plasmid sticky ends.

3. DNA ligase is added. DNA ligase catalyzes the covalent linkage of the β-globin gene fragment to the plasmid DNA molecule.  Ligation produces a population of circular DNA molecules, called recombinant DNA molecules, in which an insert is contained within the plasmid.  Some of these recombinant DNA molecules contain the β-globin gene insert.

4. Transformation of a host 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 cell via transformation. 
    
5. Host bacteria are grown on a medium that contains ampicillin. Growth produces colonies of bacterial cells, with each cell containing many copies of the recombinant DNA molecules.  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 they lack the amp^R gene.
  • Some E. coli cells were transformed. Since the 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 β-globin 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 in the plasmid is within a DNA sequence called lacZ.  The lacZ gene produces the enzyme β-galactosidase.  Cloning the insert disrupts the lacZ gene, allowing researchers to distinguish colonies that contain an insert versus colonies that do not contain an insert.

• • Recombinant plasmid vector without an insert. In this case, the cell contains an intact lacZ  The 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 includes a compound called IPTG, which activates the lacZ gene to produce the β-galactosidase protein, and X-Gal, which is a substrate for β-galactosidase.

• • 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 β-globin insert. All the white colonies contain an insert; however, at this point, we cannot distinguish the white colonies that contain the β-globin 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 the β-globin 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 β-globin gene indicates that the chosen colony likely contains the β-globin insert.  Alternatively, the polymerase chain reaction (PCR), using primers specific for β-globin can be used to amplify the β-globin insert.  If a PCR product is produced using these β-globin primers, then the β-globin gene was cloned successfully.  Finally, determining the nucleotide sequence of the cloned insert by DNA sequencing will determine if the recombinant molecule contains the β-globin gene.