Unit 4 DNA technology

• Cloning genes

– into bacteria

– Cloning genes into plants

– Cloning genes into animals

• Cloning animals (reproductive cloning)

• Stem cell research

– Therapeutic cloning

I. Gene Cloning, recombinant DNA

Cloning genes into bacteria

• Examples: GFP, Human growth hormone

Human Growth Hormone (hGH) cloned into bacteria (1980s)

• Pre-1980s

hGH purified from cadaver brains

Drawbacks?

• Today

– hGH has been cloned 26 inches tall

Cloning a gene into bacteria

1. Isolate DNA, cut hGH gene out with restriction enzymes

….ggattgcgtacgctttgatcgtagtaataggacctagtgtgtacgtaagcgg..

….ccattcgcatgcgaaagtagcatcattatccaggatctcacatgcattcgcc..

2. Obtain pure hGH gene gatcgtagtaatagg

agtagcatcattatcc

3. Ligate hGH gene into plasmid vector

Ori, ampr, restriction sites for cloning, small (high copy number)

Ligase enzyme requires ATP

this is recombinant DNA (fig 8.3)

4. transform bacteria

5. Grow bacteria = the hGH gene is now cloned

billions of copies overnight

bacteria will express the hGH gene make hGH

protein

6. Bottle, sell, and inject before puberty

• Advantages:

Other cloned drugs

Human insulin 1987

Factor VIII for hemophiliacs 1993

Interferon for chemotherapy 1993

EPO for anemia 1992

FSH for fertility clinics 1996

TPA to prevent blood clots 1996

Many drugs for domestic animals

cancer, arthritis, emphysema and other drugs

Cloning into plants (GM)

• Transgenic plants

• Inject gene into plant embryo (or plasmids can be used)

AGM con video UFO TV

few genetically modified plants

• GM con video UFO TV

Benefits Drawbacks

• Increased crop yield

• Resistance to drought, freezing increased seed costs

• Decreased use of pesticides pesticide resistant bugs

• Decreased use of herbicides resistant weeds

• Increased nutrition new allergens

• Increased shelf life may spread to other plants

• Can remove allergens harmful to insects?

Bt corn

• Corn plant engineered with gene that codes for a protein lethal to the corn borer

• soybeans resistant to herbicide

Non engineered cotton Bt cotton

Effect of insect infestation on cotton

http://cls.casa.colostate.edu/TransgenicCrops/what.html

Mustard plant genetically engineered to remove selenium from soil

Can then grind and use in selenium deficient soils

Golden Rice

• Many in world are deficient in Vitamin A

– Leading cause of childhood blindness (500,000 new cases per year)

• Rice engineered to produce vitamin A!

Controversial…….

• Do we need legislation for labeling of GM foods?

• Should GM genes, plants, animals, be patented?

Cloning genes into animals

Transgenic goats

Produce human protein (drug) in milk

Transgenic animals to produce human protein in milk

1. Isolate human EPO gene (for rbc production)

2. Ligate to tissue-specific promoter

– Promoter ONLY active in mammary gland protein only made in milk

1. Inject gene construct into animal embryo (fertilized egg)

2. Implant embryo into surrogate mother -> kid is born

• How can we get the transgenic kid to produce human drug?

3. Easy to purify from milk

• One herd can supply the world’s need of a particular drug (protein)

• Clean, disease free, easy to produce

Pail of milk with EPO Bottled EPO drug

Other proteins made in transgenic sheep and goat milk

• TPA – anti clotting

• Spider silk (BioSteel) – The dragline form of spider silk is regarded as the

strongest material known; it's 5 times stronger than steel and twice as strong as Kevlar.

• Anti HIV protein

• Anticancer drugs

• Alpha1-antitrypsin for emphysema

Drag line spider silk in goat milk

• The common garden spider (genus Araneus) produces spider silk light weight, strong!

• Medical devices, military, inducstry

• These mice are models for human disease (Alzheimer)

• This mouse is genetically modified to be diabetic

Agriculture

• This pig is genetically engineered to be able to digest more and produce less manure

• Other pigs produce meat high in omega 3 fatty acids

Xenotransplantation

• This pig is genetically engineered to have an immune system similar to humans’

Fish farming

• genetically engineered salmon grow faster

Patenting

• Raw products of nature are not patentable.

• DNA products become patentable when they have been isolated, purified, or modified to produce a unique form not found in nature.

• Millions of patents

Cloning beef

http://www.pbs.org/wgbh/nova/sciencenow/3302/05.html

3 types of cloning

• 1. gene cloning

–Recombinant bacteria (as in lab)

– Transgenic animals and plants

• 2. reproductive cloning

– Yields an organism

– Embryo twinning or SCNT

• 3. therapeutic cloning

– For stem cells to treat disease

Reproductive cloning

Animals that have been cloned

SCNT = somatic cell

nuclear transfer

pg. 577

1997 Ian Wilmut

Nuclear transfer

Somatic cell nuclear transfer

1. Obtain somatic cell from donor ewe 2. Serum starve to induce Go

3. Place nucleus into enucleate egg

4. Grow for 6 days

5. Implant into surrogate mother

277 embryos -> 1 lamb (Dolly)

http://learn.genetics.utah.edu/units/cloning/

Our somatic nuclei (DNA from a differentiated cell) can be reprogrammed to embryonic state!

Why clone animals?

• Models for disease

• Pharming

• Endangered species

• Reproduce deceased pet

• Help infertile couples

• K.C., the first animal produced by cloning from a cell taken from a carcass, was born in April 2002.

Problems with reproductive cloning

• High failure rate ~ 0.3% success rate– Enucleate egg may not function– Embryo may not divide– Embryo may not implant– Miscarriage

• Large offspring syndrome (LOS)– With abnormally large organs that don’t function

correctly

• Abnormal gene expression– We don’t understand how the nucleus is

reprogrammed (its old DNA in a new egg!)

• Telomere problems– Older DNA has shortened telomeres, but some

clones show lengthened telomeres

Ethical implications

• Is human cloning "playing with nature?" If so, how does that compare with other reproductive technologies such as in vitro fertilization or hormone treatments?

• If a clone originates from an existing person, who is the parent?

• What are some of the social challenges a cloned child might face?

• Should cloning research be regulated? How, and by whom?

All countries have banned human reproductive cloning.

Therapeutic cloning to treat diabetes and other disorders

• http://www.pbs.org/wgbh/nova/sciencenow/video/3209/r04-220.html

• Make embryonic stem (ES) cells

•

1. Isolate nucleus from a somatic cell – which?

2. Remove egg nucleus from donated egg

Somatic cell nuclear transfer

Therapeutic cloning

How many chromosomes in

nucleus of somatic cell?

• 3. inject somatic cell nucleus into enucleate egg

http://www.research.uci.edu/tmf/dnaMicro.htm

4. Grow to blastocyst stage

3 day embryo (morula) 5 day blastocyst

How many times has the fertilized egg divided?

Cells at this stage are totipotent and undiffferentiated

Blastocyst ~ 100 cells, day 4Hollow ball of cells with inner cell mass

2 types of cells: T -> placenta, ICM -> embryo

5. Take inner cell mass, transfer to flask, and ES cells will reproduce.

How do we get the cells to differentiate into what we want?

~100 cells

Stem cells

Questions

• Sperm?

• Fertilization?

• Embryo?

Embryonic stem cells (ES)

• not differentiated

– can become any tissue - muscle, cartilage, bone, nervous….. = pluripotent

– can grow in culture (petri dish)

Types of stem cells (handout) Totipotent stem cells (ES) can differentiate into any cell

type including placenta Example: fertilized egg , early embryo

Pluripotent stem cells (ES) - develop 4 days after fertilization, blastocyst can differentiate into any cell type (but not totipotent)

Multipotent stem cells can give rise to a number of cell types example: hematopoietic stem cells in the bone

marrow

Progenitor cells - produce only 1 cell type. Example, erythroid progenitor cells differentiate

into only rbc.

Sources of stem cells

Therapeutic cloning

Advantage = no immune rejection

Not dependent on transplant from another person

Left over in vitro fertilization 800,000 unused embryos currently frozen

Donated sperm and eggs

Umbilical cord blood, placental blood, bone marrow

Therapeutic cloning is not reproductive cloning

Reproductive cloning -> Implant into female (uterus)->- birth

ILLEGAL, rarely successful in animals

Therapeutic cloning

Place cells into culture dish

Cells divide to produce more ES cells

Use to treat /cure disease

ES cells/embryo

Uses of ES cells

1. tissue transplants – new liver cells, pancreas cells

2. Replace lost cells: Alzheimer disease, spinal cord injury, Parkinson’s disease, multiple sclerosis, diabetes, burned tissue, stroke, lung disease, heart disease, arthritis

NOTE – ES cells cannot develop into a fetus – why?

Other advantages of stem cells

Not limited by organ donation

– 80,000 people in the US are waiting for a transplant

– Over 5,000 people die each year waiting

Legal aspects of cloning: 15 states have laws

Arkansas, Indiana, Iowa, Michigan, North Dakota and South Dakota prohibit therapeutic cloning

CA, CT, MD, MA, NJ, RI allow research on embryos (restrictions vary)

New Jersey Permits human cloning for stem cell research; prohibits reproductive cloning, which is punishable as a crime in the first degree; prohibits sale or purchase, but not donation, of embryonic or fetal tissue, which is punishable as a crime in the third degree and a fine of up to $50,000

Source: http://www.ncsl.org/programs/health/genetics/embfet.htm

• A woman cannot have biological children and would like one. Her eggs are mixed with her husband’s sperm in a dish and a resulting embryo is implanted

• A woman cannot have biological children and would like one. One of her nuclei is mixed in a dish with an enucleate egg and a resulting embryo is implanted

• A person with diabetes is near death due to kidney failure. Even though he took insulin throughout life, the toll of the disease on his organs is critical. He is on a waiting list for a kidney transplant.

• A person with diabetes uses one of his nuclei to make stem cells which are induced to form pancreatic cells in a Petri dish. His diabetes is cured at age 5.

Bonnie bassler quorum sensing

optional video

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Genome = all the genetic information in a cell

• Human Genome Project – sequence human DNA

• initiated in 1990

Goals

• identify all the ~20,000 genes in human DNA

• determine the sequences of the 3 billion nucleotide base pairs in human DNA

• store this information in databases

– Available on Internet to everyone• improve tools for data analysis,

• transfer related technologies to the private sector, and

• address the ethical, legal, and social issues (ELSI) that may arise from the

project.

Accomplished by:

• Cut the genome into small pieces (150,000 bp)

• Clone into bacteria

• Sequence small sections at a time

• Assemble the information

• Government group

• Craig Venter (Celera) private

FYI

• much of the DNA for the public HGP came from a single anonymous male donor from Buffalo, New York (code name RP11).[

• Celera used 5 individuals, one of which was Venter

• >95% of the genome is completed

• There are rice, dog, corn, mosquito, horse and many more genome projects underway

Dideoxy DNA sequencing

• DNA, primer (1), G,A,T, C, + 4 chain terminators, buffer, radioactive tracer nucleotide polymerase in 4 tubes

• Electrophorese on large gel

• Transfer gel to filter paper, dry, and expose to film

• DNA ladder is observed, each band differs by one base

Tumor gene autoradiograph of sequencing gel

G A T C

DNA sequencing

Use one tube with fluorescently labeled ddNTPS and run through detector

Functional genomics

• Genome – all DNA in an organism’s chromosome(s)

• Transcriptome – all mRNA transcripts in a cell

Pharmacogenomics (handout)

• an individual's genetic inheritance affects the body's response to drugs

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DNA polymorphisms (ch. 9 pg. 213)

• People differ in nucleotide sequence

• Genes

• Non-coding sequences

• Differences can be used as DNA markers

• To identify individuals

• To diagnose disease or predisposition to disease

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SNPs = Single nucleotide polymorphisms(handout)• DNA sequence variations that occur when a single

nucleotide in the genome sequence is altered

• Occur ~1/300 bases in human genome– Each person has about 30 new ones

– Inherit others

• Make up ~ 90% of all human genetic variation

• Effort underway to map all human SNPs (~3 million)

77What is the size of the human genome in base pairs?

• Many SNPs have no effect on cell function (silent)

– Non-coding region ?

– Coding region ?

• Others could:

– cause disease

– influence disease progression

– predispose to disease

– influence response to a drug

78

SNP in tumor (melanoma)

Melanoma C to T base change at position 11701 (intron1) compared with control PBLs.

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SNP

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Alcohol dehydrogenase SNPs

• Some SNPS in this gene associated with extreme sensitivity to even small doses of alcohol

– tachycardia, vomiting

– high incidence in those of Asian descent

– 13 SNPs found in human population

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A few SNPs in the ALDH gene

• ALDH1B1*1 Wild type

• ALDH1B1*2 257 C=>T

• ALDH1B1*3 320 T=>G

• ALDH1B1*4 183 C=>T

• ALDH2*1 Wild type

• ALDH2*2 1510 G=>A

• ALDH2*3 1486 G=>A

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Goals of pharmacogenomics

1. SNP maps will identify multiple genes associated with complex diseases such as cancer, diabetes, vascular disease, mental

illness etc…

Association study = SNP pattern associated with disease

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2. Find better drugs

• People inherit/exhibit differences in drug:– Absorption

– Metabolism and degradation of the drug

– Transport of drug to the target molecule

– Excretion of the degradation products

100,000 deaths and 2.2 million adverse reactions reported each year (JAMA) = Drug reactions are the leading cause of hospitalizations and death in US

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• More specific, safer drugs

• Get it right the first time

• Dosage and drug specific to individual

• Create individual metabolic profile

• Screening– presymptomatic testing

– carriers

– preimplantation, prenatal, newborn screening

– disease susceptibility

85

Will be commonplace in the doctors office

– Genetic ID cards?

– Certain disease have gene expression “signatures”

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