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)

A GM con video UFO TV few genetically modified plants • GM con video UFO TV

Benefits • • • • • • •

Increased crop yield Resistance to drought, freezing Decreased use of pesticides Decreased use of herbicides Increased nutrition Increased shelf life Can remove allergens

Drawbacks increased seed costs pesticide resistant bugs resistant weeds new allergens may spread to other plants harmful to insects?

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

Effect of insect infestation on cotton

Non engineered cotton

Bt 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/3302/0 5.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

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

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)

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

Therapeutic cloning

• Make embryonic stem (ES) cells • 1. Isolate nucleus from a somatic cell – which? 2. Remove egg nucleus from donated egg

How many chromosomes in nucleus of somatic cell?

 Somatic cell nuclear transfer

• 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 4 Hollow 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.

~100 cells

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

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 ES cells/embryo

Therapeutic cloning Reproductive cloning -> Implant into female (uterus)->- birth ILLEGAL, rarely successful in animals

Place cells into culture dish Cells divide to produce more ES cells

Use to treat /cure disease

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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• Human Genome Project – sequence human DNA • initiated in 1990

Genome = all the genetic information in a cell

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)

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

• 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

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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 ALDH1B1*2 ALDH1B1*3 ALDH1B1*4 ALDH2*1 ALDH2*2 ALDH2*3

Wild type 257 C=>T 320 T=>G 183 C=>T Wild type 1510 G=>A 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 84

• • • • •

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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