chapter 20
1. 2 basic purposes of gene cloning
To make any copies of, or amplify, a particular gene
To produce a protein product
2. concepts of cloning vector and recombinant DNA
SLIDE 34
A cloning vector is a DNA molecule used to carry foreign DNA into a host cell for replication and expression. It is a critical tool in genetic engineering.
Key Features of Cloning Vectors:
1. Origin of Replication (Ori):
o Ensures the vector replicates within the host cell.
2. Selectable Marker Genes:
o Allow identification of cells that have taken up the vector (e.g., antibiotic resistance genes).
3. Multiple Cloning Site (MCS):
o A region with several unique restriction enzyme sites for inserting foreign DNA.
4. Size and Stability:
o Small enough for efficient transfer and stable replication in host cells.
Examples of Cloning Vectors:
· Plasmids: Circular DNA found in bacteria.
· Bacteriophages: Viruses that infect bacteria.
· Cosmids: Hybrid of plasmids and bacteriophage λ.
· Artificial Chromosomes: BACs (bacterial artificial chromosomes) and YACs (yeast artificial chromosomes).
Recombinant DNA
Recombinant DNA (rDNA) is DNA formed by joining genetic material from two different sources, often from different species, to create a new combination of genes.
Steps to Create Recombinant DNA:
1. Isolation of DNA:
o The desired gene and the vector are isolated.
2. Cutting DNA:
o Restriction enzymes (molecular scissors) cut the DNA at specific sequences.
3. Ligation:
o DNA ligase joins the foreign DNA with the vector at compatible sticky or blunt ends.
4. Transformation:
o The recombinant DNA is introduced into a host organism (e.g., E. coli).
5. Selection and Screening:
o Only host cells with recombinant DNA are identified and grown.
Applications of Recombinant DNA:
· Producing insulin and other pharmaceuticals.
· Creating genetically modified organisms (GMOs).
· Gene therapy for treating genetic disorders.
3. While constructing a plasmid or inserting a gene into a plasmid, in practice, the vector/plasmid and the insert should be cut with the same set of restriction enzymes to produce compatible ends which in turn would facilitate the ligation later.
4. 2 Slides on PCR
Kary Mullis, 1983
PCR: Polymerase Chain Reaction, to amplify DNA
Things needed: DNA template, DNA polymerase, dNTPs, primers.
Taq DNA polymerase: thermal state, isolated from the bacterium, Thermus aquaticus
It can produce many copies of a specific target segment of DNA
The key to PCR is an unusual, heat-stable DNA polymerase called Taq polymerase. Other polymerases may be used as well; some are more accurate and stable than Taq, such as Pfu polymerase
PCR uses a pair of primers specific for the sequence to be amplified
PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
5. concept of totipotent
In plants, mature cells can “dedifferentiate” and then
give rise to all the specialized cell types of the organism
• A totipotent cell, such as this, is one that can generate a
complete new organism
• Plant cloning is used extensively in agriculture
6. slide on “stem cells”
Stem cells are relatively unspecialized cells that can both
reproduce indefinitely and, under certain conditions,
differentiate into one or more specialized cell types
Many early embryos contain stem cells capable
of giving rise to differentiated embryonic cells of
any type
• In culture, these embryonic stem (ES) cells reproduce
indefinitely, and depending on culture conditions, can be
made to differentiate into a variety of specialized cells
• Adult stem cells can generate multiple (but not all) cell
types and are used in the body to replace nonreproducing
cells as needed
7. slide on “working with stem cells”
SLIDE 48
8. concept of induced pluripotent stem cells
Induced pluripotent stem cells (iPSCs) are specialized cells that have been genetically reprogrammed to behave like embryonic stem cells, meaning they can differentiate into almost any cell type in the body (pluripotency).
Key Concepts:
1. Derivation:
o iPSCs are generated by taking somatic (adult) cells (e.g., skin or blood cells) and introducing specific genes that reprogram them into a pluripotent state.
o The most common reprogramming factors are the Yamanaka factors: Oct4, Sox2, Klf4, and c-Myc.
2. Pluripotency:
o Like embryonic stem cells, iPSCs can give rise to the three germ layers:
§ Ectoderm: Skin, neurons.
§ Mesoderm: Bone, muscle.
§ Endoderm: Liver, lungs.
3. Advantages:
o Avoid ethical concerns associated with using embryonic stem cells.
o Potential for personalized medicine, as iPSCs can be derived from a patient’s own cells, reducing the risk of immune rejection.
4. Applications:
o Regenerative Medicine: Repairing damaged tissues or organs.
o Disease Modeling: Studying diseases in lab-generated patient-specific cells.
o Drug Testing: Testing the effects of drugs on human cell types.
o Gene Therapy: Correcting genetic defects by reprogramming and editing cells.
5. Limitations:
o Risk of tumor formation due to genetic modifications (e.g., c-Myc is an oncogene).
o Incomplete reprogramming or low efficiency in creating iPSCs.
Shinya Yamanaka received nobel prize in 2012in medicine for this work with John Gurdon
9. concept of gene therapy
Gene therapy is the introduction of genes into an afflicted
individual for therapeutic purposes
• It holds great potential for treating disorders traceable to a
single defective gene
• Bone marrow cells are prime candidates for gene therapy
because they multiply throughout the patient’s life
• Gene therapy and gene editing provoke ethical questions
• Some critics believe than any tampering with human genes
is unethical
• Others see no difference between transplanting genes into
human cells and transplanting organs between people
• Jennifer Doudna, a co-discoverer of CRISPR-Cas9,
recognized not only its potential but also the danger of its
misapplication
