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Cloning vectors
Common properties
origin of DNA replication
unique restriction sites for insertion of DNA
multiple cloning sites containing many restriction sites engineered into many
plasmid vectors
Types of vectors
plasmids containing drug resistance gene (up to 10 kb)
many commercially available plasmids
Restriction Maps
Restriction Maps
Line drawings of DNA identifying sites cut by
restriction endonucleases.
Identify potential RFLP markers for genetic
diagnosis.
Example: Restriction site polymorphism for Mstll
may be used to identify individuals with the sickle cell
mutation
Expression Vectors
If the goal of the cloning procedure is to obtain a
recombinant protein, appropriate sequences required
for transcription and translation in the cloning host
cell must be provided because they will not be part of
the cDNA.
For instance, to produce recombinant human insulin
in bacteria, a bacterial promoter and a ShineDalgarno sequence must be included in the cloning
plasmid near the insertion site for the cDNA.
Figure 1-6-5 shows an example of an expression
vector, pUC. In some expression vectors, other
regulatory sequences such as operators are added to
allow expression of the cloned gene to be controlled.
Transgenic Animals
Transgenic animals are produced by transferring cDNA into
the pronucleus of a fertilized ovum. The resultant transgenic
animal has the new gene (transgene) in all of its cells including
its reproductive tissues. Transgenic animals are now widely
used as experimental models in which to study human diseases.
A variation of this technique produces a knockout animal, in
which a normal gene has been functionally eliminated. This
may be done by site-specific mutagenesis.
Transgenic animals have a new gene (transgene) introduced
into their germline.
All cells of a transgenic animal contain the transgene.
Transgene is inherited by offspring.
Knockout animals have a normal gene intentionally
inactivated/destroyed.
Transgenic and knockout animals are used as models of human
disease.
Gene Therapy
When using gene therapy to correct genetic deficiencies in
humans, the cloned normal gene is targeted only to the tissues
giving rise to the major symptoms.
For instance about 50% of the cases of severe combined
immunodeficiency (SCID) are caused by mutations in the gene
for a subunit common to several interleukin receptors.
The approach to gene therapy has been to introduce a normal
cloned gene into the patient's bone marrow cells that will
subsequently divide and differentiate to produce T and B
lymphocytes expressing the gene.
These patients subsequently have improved immune function.
Because the cloned normal gene has not been introduced into
reproductive tissues any children of the patient would inherit
the defective allele.
Review Questions
Select the ONE best answer.
1. If a cystic fibrosis patient were to be treated by
gene therapy, which type of cells should be
targeted as host cells?
A. Germ cells
B. Epithelial cells
C. T cells
D. Hemopoietic stem cells