PART 1:DNA Cloning and Plasmids

DNA Technology and Genomics

Biotechnology

• DNA technology has launched a revolution in the area of biotechnology–The manipulation of organisms or their

genetic components to make useful products• Example: gene cloning

»Usually uses bacterial plasmids

Plasmid• A circular, double-stranded unit of DNA that replicates within a cell independently

of the chromosomal DNA. Plasmids are most often found in bacteria and are used in recombinant DNA research to transfer genes between cells.

Figure 20.2

Bacterium

Bacterialchromosome

Plasmid

Cell containing geneof interest

RecombinantDNA (plasmid)

Gene of interest

DNA ofchromosome

Recombinantbacterium

Protein harvested

Basic research on protein

Gene of interest

Copies of gene

Basic research on gene

Gene for pestresistance inserted into plants

Gene used to alterbacteria for cleaningup toxic waste

Protein dissolvesblood clots in heartattack therapy

Human growth hormone treatsstunted growth

Protein expressedby gene of interest

3• Overview of

gene cloning with a bacterial plasmid, showing various uses of cloned genes

How do You Make Recombinant DNA?

• Use bacterial restriction enzymes– Cut DNA molecules at a limited number of specific

DNA sequences, called restriction sites

Recombinant

Plasmid

Restriction Enzymes: Proteins that cut the DNA in a specific place

Transformation bacteria takes in plasmid from environment

There are 3 main reasons for creating recombinant DNA 1.to create a protein product2.to create multiple copies of genes3.to insert foreign genes into other organisms to give those organisms a new trait4.Make DNA fingerprints

•Recombinant DNA is used widely today to create large amounts of protein for treating illnesses• Ex: In 1982, insulin became the first recombinant DNA

drug to hit the market

Why make DNA recombinants?

The DNA of plants and animals is altered for several reasons

PLANTS1. Disease-resistant 2. Insect-resistant crops3. Hardier and bigger fruit 4. Increased vitamin content5. “Pharming” purposes (ex.

Vaccine)

70-75% of food in supermarket is genetically modified.

Genetically modified organisms are called transgenic organisms.

TRANSGENIC ANIMALS

1. Fish – grow faster than normal aquaculture salmon

2. Chickens – more resistant to infections

3. Cows – increase milk supply and leaner meat 4. Goats, sheep and pigs – produce human proteins in their milk

What are Genetically Modified Foods?

(“Frankenfoods”?)

You have already eaten GM foods. Some GM tomatoes, for example, have had their genes altered to stop them from going soft while they are still growing. For several years they were widely sold in tomato paste.

The GM foods we eat have all been tested for safety. But some people worry about the long term effects of eating genetically modified foods!

'Frankenfood' FearsExperts say this science, like any other, has no guarantees. Risks

include:• Introducing allergens and toxins to food• Accidental contamination between genetically modified and non-

genetically modified foods• Antibiotic resistance• Adversely changing the nutrient content of a crop• Creation of "super" weeds and other environmental risksBenefits include:• Increased pest and disease resistance• Drought tolerance• Increased food supply

Can We End World Hunger? Malnutrition and vitamin deficiencies?

Golden rice is genetically modified rice that now contains a large amount of A-vitamins. Or more correctly, the rice contains the element beta-carotene which is converted in the body into Vitamin-A. So when you eat golden rice, you get more vitamin A.

Advantages: • The rice can be considered a particular advantage to poor people in underdeveloped

countries. They eat only an extremely limited diet lacking in the essential bodily vitamins. The consequences of this restricted diet causes many people to die or become blind. This is particularly true in areas of Asia, where most of the population live on rice from morning to evening.

Going Bananas? According to recent reports, the world may soon be out of bananas.

Bananas are propagated through asexual cloning, so all bananas are genetically identical

Banana plantations in Africa, Asia and Central America are uniquely susceptible to fungi, viruses and pests.

Unless scientists can find a way to genetically enhance the banana’s ability to ward off parasites, we could be bananaless in ten years.

http://gslc.genetics.utah.edu/features/gmfoods/

Examples• For example, taking a gene from a

jellyfish and inserting into the zebrafish genome created the “glofish” – fish that glow in the dark which are now being sold on the market.

• Another example is genes from a bacteria inserted into food crops so that the crop can create it’s own insecticide. These plants and animals are also known as “transgenic”.

Human DNA in a Goat Cell

This goat contains a human gene that codes for a blood clotting agent. The blood clotting agent can be harvested in the goat’s milk. .

Transgenic Goat

Part 2: The Genetic Basis of Development:

Stem Cells and Cloning

DNA Technology and Genomics

Model Organisms

• used understand broad biological principles

DROSOPHILA MELANOGASTER(FRUIT FLY)

CAENORHABDITIS ELEGANS(NEMATODE)

0.25 mm

Small, easily grown, short generation time, embryos outside body, 13,700 genes

Small, easily grown, short generation time, transparent, 19,00 genes

MUS MUSCULUS(MOUSE)

DANIO RERIO(ZEBRAFISH)

ARABIDOPSIS THAMANA(COMMON WALL CRESS)

Other “model organisms”

Small, easily grown, body, 25,000 genes

Embryonic development

• Embryonic development involves–cell division (a.k.a.- mitosis), –cell differentiation

• In embryonic development of most organisms–A single-celled zygote gives rise to cells of

many different types, each with a different structure and corresponding function

3 Interrelated Processes…• Through a succession of mitotic cell

divisions–zygote gives rise to large # of cells

• In cell differentiation–Cells become specialized in structure and

function

Specialized cells perform specific functions

• Cell differentiation is the process by which unspecialized cells develop into their mature forms and functions.

• Your body began as a single fertilized egg. • While almost every cell in your body has a full set

of DNA, each type of cell uses only the specific genes it needs to carry out its function. That is, a cell differentiates among the genes and uses only certain ones– Cookbook has recipe for dessert, pasta, etc but same

cookbook.

CELLS...Totipotency• A totipotent cell–cell capable of generating a

complete new organism• Cloning–using 1+ body cells from a

multicellular organism to make another genetically identical individual

Nuclear Transplantation in Animals

• In nuclear transplantation (nuclear transfer)–nucleus of unfertilized egg cell or

zygote is replaced with nucleus of a differentiated cell

Reproductive Cloning of Mammals

• In 1997, Scottish researchers–1st cloned ANIMAL a lamb from an adult sheep

by nuclear transplantation–Named “Dolly”–Aged quickly and didn’t live long.–Possibly because she was cloned from an adult

sheep that had “old DNA”.–Died at age 6 of complications that usually affect

older sheep• Incomplete reprogramming of original nucleus?

Mammalian Cloning

Lamb (“Dolly”)genetically identical to

mammary cell donor

“Copy Cat”• Was the first cat ever cloned• Clones in same species do NOT necessarily

look or behave alike epigenetics

Problems With Cloning• In most nuclear transplantation studies –Only a small % of cloned embryos

develop normally–Most (all?) have various defects if

clone lives–It takes hundreds of tries to produce

one clone

The Stem Cells of Animals ONLY

• A stem cell (topipotent cells) –A relatively unspecialized cell –Can reproduce itself indefinitely–Can differentiate into specialized cells of one

or more types, given appropriate conditions–2 types: embryonic & adult

Stem Cell Classification

• Totipotent stem cells can grow into any other cell type. Only a fertilized egg and the cells produced by the first few divisions of an embryo are totipotent.

• Pluripotent stem cells can grow into any cell type except for totipotent stem cells.

• Multipotent stem cells can only grow into cells of a closely related cell family.

Stem Cell ClassificationClass Totipotent Pluripotent multipotent

Type of Cell Fertilized egg Embryonic stem cell Adult stem cell (example from blood)

Can give rise to

All cells Almost any cell Closely related cells

Example New organism Neurons, skin, muscle, kidney, cartilage, bone, liver, pancrease

Red blood cells, platelets, white blood cells.

Adult stem cells–Said to be pluripotent•able to give rise to multiple but not all cell

types

Embryonic stem cells–Said to be topipotent•able to give rise to all cell types

Early human embryoat blastocyst stage

(mammalian equiva-lent of blastula)

From bone marrowin this example

Totipotentcells Pluripotent

cellsCulturedstem cellsDifferentculture

conditions

Differenttypes of

Differentiated cells

Liver cells Nerve cells Blood cells

Embryonic stem cells

Adult stem cells

• Stem cells can be isolated – From early embryos at the blastocyst stage