DNA Cloning & Gene Therapy

Syed Ahmad Ali

Mc 3847
 DNA Cloning
 Technique to prepare multiple copies of specific sequences
of nucleotides
 Production of protein product
 Production of many copies of gene itself
 Restriction Endonucleases

 Bacterial enzymes used to cleave foreign DNA

 Specificity
 Sticky and Blunt ends
 Restriction Fragments
 Recombinant DNA vectors
 DNA molecule to which gene of interest is joined
 Characteristics

 Antibiotic resistant gene

 Capacity of autonomous replication.
 Nucleotide sequences recognized by restriction endonucleases
 DNA Libraries

Genomic DNA library cDNA Library

 Every DNA sequence
 Introns and exons both are
present
 Only those sequences that
appear processed as
mRNA
 Only exons are present
 What Is Gene Therapy?
 An approach of treating disease by modifying expression of
gene or correction of abnormal gene
 Knocking out mutated gene
 Introduction of normal gene
 Replacement of mutated gene
 TYPES OF GENE THERAPY

Somatic Gene Therapy Germ line Gene therapy

 Therapeutic gene transferred into  Therapeutic gene transferred into
Somatic cell germ line cells
 Not inherited  Inherited
 Blood cells,skin cells  Sperms and eggs
 Example Of In Vivo Gene Therapy
 Cystic fibrosis
 A protein called cystic fibrosis trans-membrane regulator
(CFTR) is absent due to CFTR gene defect
 Treated by in vivo replacement of defective gene by
adenovirus vector.
 Example Of Ex Vivo Gene Therapy
 1st gene therapy - to correct deficiency of enzyme Adenosine
deaminase (ADA)
 Performed on a 4yr old girl Ashanthi DeSilva
 Was suffering from SCID
 Caused due to defect in gene coding for ADA
 Deoxyadenosine accumulate and destroys T lymphocytes
 Advantages
 Gene therapy has the potential to eliminate and prevent
hereditary diseases such as cystic fibrosis, ADA,SCID etc.
 It is a possible cure for heart disease, AIDS and cancer
 It gives someone born with a genetic disease a chance to
life
 It can be used to eradicate diseases from the future
generations.
 Disadvantages
 Long lasting therapy is not achieved by gene therapy
 Due to rapid dividing of cells benefits of gene therapy is short lived
 Immune response to the transferred gene stimulates a potential risk
to gene therapy
 Disorders caused by defects in multiple genes cannot be treated
effectively using gene therapy
 Viruses used as vectors for gene transfer may cause toxicity, immune
responses, and inflammatory reactions in the host.
Any Questions?
Thank
You