Recombinant DNA TechnologyIntroduction to Cloning

Chromewell MojicaNational Institute of Molecular Biology and Biotechnology

University of the Philippines, Diliman Quezon City

At the end of the session, the students should:

1. Be able to understand recombinant DNA technology

2. Be able to define Cloning and the 3 types of cloning

3. Be able to know the different products of recombinant DNA technology

Session Objectives

“Genetic manipulation”

“Genetic modification”

“Recombinant DNA technology”

The technique of altering the genetic makeup of cells or organisms by deliberately inserting, removing, or

altering individual genes

What is Genetic Engineering?

Any organism whose genetic material has been altered.

• Contains a foreign piece of DNA• Deleted some portions of the Genome• Mutated genome

Genetically Modified Organisms (GMOs)

1. Improves crops and livestock2. Massive production of industrially important

enzymes/proteins3. Medical purposes: therapeutic cloning, cheaper

medicine4. Bioremediation5. And MANY more! ☺

The need for Genetic Engineering?

A DNA sequence produced artificially by joining pieces of DNA from different organisms

Created by “cutting and pasting” different DNA fragments

Recombinant DNA (rDNA)

DNA fragment containing the gene ofinterest (transgene)

Plasmid DNA withSelective marker

Restriction enzyme

Ligase

Recombinant DNA (rDNA): The Ingredients

RE: Enzymes that cut DNA in specific sequence

Restriction Enzymes

RE: Enzymes that cut DNA in specific sequence

Recombinant DNA (rDNA): The Ingredients

Production of any organism with the same genetic replica of another cell, tissue or organism

Clone: The organism bearing the copied material

What is Cloning? A clone?

Three Types of Cloning

Similar to recombinant DNA technology

Only certain parts of DNA is copied andtransferred from one organism to another

Uses self replicating PLASMID as VECTOR

DNA/Gene Cloning

Transformation (transfection):Introduction of foreign DNA into cells

Modes of transformation/transfectionElectroporation: Electrical pulseHeat shock: Brief exposure to 37°C-45°CGene gun (biolistics): DNA-coated beads (bullets)Liposome-mediatedViral vectorsInjection of DNA into nucleus

Transformation

Selection and Screening of Successful Transformants

Selection and Screening of Successful Transformants

GM Bacteria for Human Insulin Production

Producing GM Plant

Crown Gall Diseasein Plant

Producing GM Plant

GM Bacteria for Human Insulin Production

Non-Bt corn Bt corn

Three Types of Cloning

Production of a genetic duplicate of an existing organism.

Accomplished using a process called somatic cell nuclear transfer (SCNT)

involves putting the nucleus of a body (somatic) cell into an egg cell from which the nucleus has been removed. (enucleated oocyte)

Reproductive Cloning

Somatic Cell Nuclear Transfer

Other cloned animals via SCNT

Somatic Cell Nuclear Transfer

Three Types of Cloning

Like reproductive cloning but the embryo is not allowed to fully develop

Main purpose is to generate embryonic stem cells (ESC) for therapeutic purposes.

Therapeutic Cloning

STEM CELLS

Mass of undifferentiatedcells that may give rise to different types of cells

Reproductive Cloning

Uses of Stem Cells

Can be used to replace dead cells or damage cells

Can be used to generate specific tissues and organs if provided with specific growth factors

END.

QUESTIONS?