Biology - Cloning/Stem Cell Research, and DNA Technology

Cloning definition

Creating genetically identical cells

Cloning types

1. Therapeutic cloning.

2. Reproductive cloning

Nuclear Transfer

Introduction of a nucleus from a body cell into an egg cell (with it's nucleus removed) to make an embryonic stem cell and later generate an organ or organism identical to the nucleus donor

Nuclear Transfer Example

Used in the making of Dolly the sheep

• Attempted approx. 300 times and only worked once (Now 10-20% success rate)

• Dolly had premature aging and died early

Results of therapeutic cloning

New parts of an organism (transplants) but cells must be signaled to become a specific type of cell using growth factors or chemicals

Results of reproductive cloning

The creation of a new organism

Why is reproductive cloning used and what is it used on?

• Useful for mass producing desirable farm animals for food or pharmaceutical drugs (have useful genes for wanted proteins) (Ex: Beef Cows)

• Used by people who want to clone their pets

• Sheep, mice, rabbits, horses, pigs, cats, monkeys have been successfully cloned

Stem cell research involvement

Cloning involves the making or using of stem cells

Stem cells

unspecialized cells that give rise to the different types of cells that make up the body

The two types of stem cells

1. Embryonic stem cells

2. Adult stem cells

Embryonic stem cells capability

Have the ability to become any cell type (pluripotent)

Controversy of embryonic stem cells

Some believe it is unethical to destroy embryos that have the potential to develop into babies

Traditional source of embryonic stem cells and drawbacks

• Traditionally embryonic stem cells have been obtained from unused human embryos from fertility clinics

• If organs are grown from these there would be a compatibility issue since the cells are from another cell line

Adult stem cells and origin

• Stem cells that remain in the body into adulthood

• In places like bone marrow, muscle, heart, brain tissue and umbilical cords

Benefits of adult stem cells

• Using a persons own stem cells for transplants would avoid a possible immune response (Cloned organs)

• Not as controversial since an embryo is not destroyed

Problems of adult stem cells

Normally become just one or a few types of cells unlike embryonic stem cells which give rise to all of the cells of the body (multipotent)

• Ex. Bone marrow stem cells give rise only to new blood cells

How to make iPSCs (Induced Pluripotent Stem Cells)

Altering adult stem cells to become embryonic stem cells (pluripotent)

Four genes injected using viruses or an alternative technique to make them pluripotent

why are iPSCs (Induced Pluripotent Stem Cells) good?

Unlimited supply of cells with no compatibility problem

Genetic engineering

manipulating genes for practical purposes to create genetically modified organisms (GMOs) Ex. Replacing disease genes

Biotechnology

using organisms to make useful products

Ex. Bacteria that produce insulin

Ex. Corn plants that produce their own pesticide

Recombinant DNA

Splicing together DNA from two different sources

How is Recombinant DNA done?

1. Cutting DNA into pieces

2. Splice DNA together from two different organisms

Restriction enzymes/CRISPER enzymes (CAS Protein)

Bacterial enzymes that cut DNA at specific locations called recognition sequences

What can Restriction enzymes/CRISPER enzymes (CAS Protein) do?

Can be used to destroy a gene, removing a gene and open the DNA for splicing in new genes.

Where were Restriction enzymes/CRISPER enzymes found?

Restriction enzymes were discovered in bacterial cells- used to protect them against intruding DNA from other organisms or viruses (immune system weapon) by chopping up and destroys DNA from the invader

How do we splice DNA together?

Two sources cut with the same restriction enzyme so that they have complimentary “sticky ends” that connect with each other

DNA ligase

enzyme used to seal the restriction fragments together

Plasmids

Small, circular DNA molecules that replicate separately from the bacterial chromosomes

Often used for making recombinant DNA

vectors

gene carriers

Recombinant DNA Technology in Bacteria

Inserting a gene of interest into the DNA of a bacterial cell

Makes multiple copies of the inserted gene (genomic library) Or reads the gene and makes protein from the inserted gene Protein be removed from bacteria and given to people who need it

1. Makes multiple copies of the inserted gene (genomic library)

2. Or reads the gene and makes protein from the inserted gene

3. Protein be removed from bacteria and given to people who need it

ex. Insulin – protein hormone that triggers cells in the body to absorb sugar from the blood

Recombinant DNA Technology in Plants and Animals

Bacterial gene that codes for a toxic protein to insects can be inserted into the genome of a plant so that it is resistant to the insect

Genetically Modified Organism (GMO)

an animal, plant, or microbe whose DNA has been altered using genetic engineering techniques.

Genetically Modified Organism (GMO) over the years

For thousands of years, humans have used breeding methods to modify organisms. Corn, cattle, and even dogs have been selectively bred over generations to have certain desired traits. Within the last few decades, however, modern advances in biotechnology have allowed scientists to directly modify the DNA of microorganisms, crops, and animals.

Possible Problems with GMOs?

GM bacteria/plants/animals may carry genes that are hazardous to human health or the environment

1. Create resistant strains of bacteria

2. Plants that create new proteins that people are allergic to

3. GM plants transfer their genes to weeds and make “superweeds”

To combat Possible Problems with GMOs?

Several governmental agencies test and regulate GMOs for safety

PCR – Polymerase Chain Reaction

Method of making more DNA when only a small sample is available (Like at a crime scene or from an ancient organism or a virus!)

Waht is needed for PCR

Original DNA

1. Primer- short strand of complementary DNA that jump starts replication of new complementary strand

2. Supply of DNA nucleotides (ATGC)

3. DNA Polymerase-Builds new DNA strand

   1. The Multiplied DNA can then be used in a Gel electrophoresis to get a DNA fingerprint or show the presence of a gene if dyes are used

DNA Fingerprint

A pattern of bands made from noncoding DNA between genes that varies most from person to person (Short Tandem Repeats or STRs)

Steps of DNA Fingerprinting

1. Use PCR to make copies of the STR regions of individuals DNA or use restriction enzymes to cut DNA into smaller DNA fragments

2. Separate out the fragments using a technique called gel electrophoresis that separates the pieces based upon the sizes of the fragments

3. Observe banding patterns after adding radioactive probes or stains - ex. Compare DNA fingerprint from DNA left at the crime scene to the DNA of a suspect

DNA Fingerprinting uses

1. Crime scenes (samples taken to determine who may be guilty of a violent crime

2. Family relationships (to determine the parents of a child or relations to ancestors )

3. Locate a gene of interest (ex. Disease gene)

Gene Therapy

Replacing a defective gene with a normal one using viruses and CRISPER technology. (defective gene cut and new gene added to replace it)

- Into what cells are these genes for gene therapy placed?

1. Reproductive cells or early zygote (embryo) cells

2. Stem cells in bone marrow

3. Cells that are directly being affected by the defective gene

SCIDS

delivering genes to help fix the immune system

Hemophilia

inserting clotting protein genes into liver cells

Hereditary Blindness

inserting genes into retinal cells

Fat Metabolism Disorder

inserting missing enzyme gene into muscle cells

Parkinson’s

inserting dopamine gene

Cancer-

inserting herpes gene so immune system recognizes cancer cells more effectively

Sickle Cell

inserting normal hemoglobin gene into bone marrow stem cells

Human Genome Project (HGP)

Map the entire human genome by determining the entire nucleotide sequence (done)

Current HGP work

1. identifying the genes within the sequence

2. mapping the genomes of other organisms

   1. compare to ours to see how closely related we are

Knowledge gained through the HGP

1. About 20,000 genes

2. 98% of DNA doesn’t code for anything (junk DNA)

3. 99.9% of all humans DNA is the same

4. 3 million bases out of 3.3 billion that can vary from person to person

5. Repetitive DNA – sequences present in many copies in the 98% (junk DNA)

Repetitive DNA

1. Structural use at centromere

2. Protective use at ends of chromosomes called telomeres – get shorter as a person ages

3. Transposons/switches- sequences of DNA may move and turn on and off genes (caused red streaks through these Indian corn kernels)

Diabetes treatment

Human insulin made in E. coli

Growth defects

Human growth hormone made in E. coli

Cancer treatment

Interleukin -2 made in E. coli

Needed clotting factor used for hemophiliacs

Factor VIII made in mammalian cells (mammary glands of cow, sheep) - extracted from milk

Treatment for heart attacks – clears clots in the Heart capillaries

Tissue plasminogen activator (TPA) made in mammalian cells (mammary glands of cow, sheep) - extracted from milk