bacterial genomics chapter 10, page 251 - mcccblinderl/documents/unit3students09.pdfbacterial...

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Bacterial Genomics Chapter 10, page 251

First sequenced was Haemophilus influenzae

1.83 million bp

2

Escherichia coli (1997)

Located in the lower intestines of animals

Pathogenic strains (ex. E. coli 0157)

Genome

4.6 megabases

~ 4000 genes, ~88 % of genome open reading

frames

3

Single circular chromosome

http://www.sinauer.com/cooper/4e/micrographs0603.html

http://www.emc.maricopa.edu/faculty/farabee/biobk/bactchromo.gif

4

E. coli biology

Prokaryote

nucleoid region contains the chromosome

Neisseria gonorrhoeae.

5

E. coli reproduction

Bacteria reproduce by binary

fission -> Exponential growth

6

Bacterial growth

colony - visible cluster of

clones

about 1 million cells /colony

lawn – entire plate is covered,

no individual colonies visible

Growth on agar plate

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Growth of bacteria (E. coli)

Lag phase - slow or no apparent growth

Log phase –double every 20’ to 1 X 109/ml

Stationary phase

nutrient and/or oxygen limited

Cell number remains constant

Death phase

Nutrients gone, toxic products build up, cells die

8

Bacterial growth curve

9

Growth media

minimal media =only essentials

provided

Sugar (carbon source) + salts

bacteria synthesize aa, nucleotides,

vitamins

complete media

selective media

Allows one species to grow while

selecting against another

10

Solid and liquid culture

Growth in liquid media Growth on agar plate

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Phenotypes

Prototroph

can synthesize requirements from minimal

media

Auxotroph

nutritional mutant

Requires one or more supplements to grow

12

Bacterial phenotypes

Resistant to ampicillin = Ampr

Sensitivity to streptomycin = Strs

auxotroph mutant requires tryptophan = Trp-

trp-leu-thi+tetr ?

13

Bacterial mutants

Nutritional mutants

Auxotrophs that require supplement to grow

Conditional mutants

The mutation is only expressed in a certain

condition

Resistance mutant

Antibiotic resistance in bacteria

quorum sensing

http://www.pbs.org/wgbh/nova/sciencenow/34

01/04.html

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How do bacteria undergo genetic

recombination?

16

Conjugation

parasexual mating

one-way transfer of genetic information from

“male” to “female” bacteria

17

E. coli nutritional mutants

demonstrate conjugation

Mix auxotrophs – alone cannot grow on

minimal media:

Strain A met- bio- thr+ leu+

Strain B met+ bio+ thr- leu-

OBTAIN ---> a few prototrophs that grow on

minimal media:

What would the genotype of this prototroph be?

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Fig 18.2

Its rare ! 1 /10,000,000

Genetic recombination

19

Fig. 18.3 Davis U-tube showed that

conjugation requires cell/cell contact

met- bio- thr- leu-

Media can pass but cells can’t no prototrophs obtained

Show that cell-cell contact is required

filterNote the filter

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F factor (plasmid) carries DNA

from “male” to “female” bacterium

F factor

circular, episomally maintained piece of

DNA

Encodes F pilus on donor cell

Donor cell is F+

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Conjugation fig. 18.5

F+ + F- = 2F+

Steps: Pilus -> nick DNA -> transfer DNA ->

make double stranded -> break pilus

22

F factor is a plasmid

94,000 bp

Must have an origin of replication (ori) to be maintained

can transfer to other cells.

()

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Recombination (rare): Integration of F factor into

chromosome

Hfr strain fig. 18.5

25

Hfr conjugation: F factor would transfer last

• The first DNA to be transferred is the

chromosomal DNA

• Pilus is broken before F factor is transferred

• Recipient cell remains F-

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genetic recombination with Hfr

The transferred DNA may degrade or undergo homologous recombination

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Comparing an Hfr to F+ strain

F+ x F- recipients are F+

Low frequency of recombinants upon

conjugation

Hfr x F- recipients are F-

High frequency of recombinants upon

conjugation

28

Site of integration and orientation of plasmid

integration in the Hfr bacterial DNA is random

Linear transfer of genes

So, the time it takes for a particular gene to

transfer depends on where its located on the

chromosome

Hfr strains allow mapping of the E.

coli chromosome!

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Lederberg’s experiment explained

fig. 18.7

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Interrupted mating technique to

map genes on E. coli

1. Mix donor and recipient cells. Hfr strs + F- strr

2. Incubate to allow conjugation to get started

3. At time t, blend the culture in the kitchen blender. This

disrupts the cell pairs but does not break the individual

cells.

4. Plate recipient cells (use streptomycin selection – why?).

5. Screen for recombinant markers.

Elie Wollman & François Jacob

31

The mating:

Hfr H (aziRtonRlac+gal+strS)

x

F- (aziStonSlac-gal-strR)

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Fig. 18.7

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E. coli minute map = 4.7 million bp

(4377 genes) Clock face.... Gene controlling

Noon+ threonine synthesis

1 o'clock lactose degradation (lac-operon)

2 o'clock galactose -> glucose (gal-operon)

3 o'clock tryptophan synthesis (trp-operon)

5 o'clock histidine synthesis (his-operon)

7 o'clock lysine synthesis

8 o'clock streptomycin resistance

9 o'clock mannitol degradation

10 o'clock Place where chomosome synthesis begins in both directions ("OriC")

11 o'clock methionine synthesis

Noon- "F"-episome (where "F" is inserted)

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Map genes using different Hfr strains

In E. coli, four Hfr strains donate the genetic markers

shown in the order given:

STRAIN 1: QWDMT STRAIN 2: AXPTM

STRAIN 3: BNCAX STRAIN 4: BQWDM

What is the order of these markers on the circular

chromosome of the original F+?

What is the location and orientation of the F factor

integration in the bacterial chromosome?

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Transduction –phage mediated transfer

of genes into bacteria

Phage – a virus that infects bacteria

Salmonella typhimurium bacteria and P22 virus

U-tube experiment

mix 2 auxotrophs

prototrophs appear (low rate)

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Filter prevents cell contact,

transduction still occurs

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Viral infection

1. Virus adsorbs to cell and injects DNA

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2. normal bacterial activity is shut down

and bacterium becomes a “phage

factory”

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3. host DNA broken into pieces, new

viruses released to infect new cells

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chromosomal DNA is chopped as

viruses destroy cell

Faulty head stuffing

As chromosomal DNA is broken, a piece can

get packaged into a virus.

This virus can infect a new cell and transfer

genes from the first bacterium

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Gene therapy with virus Ch 9, pg 231

Objective : insert a normal gene into human

DNA that contains mutant gene

Use virus as vector

45

Remove viral replication genes (can no

longer cause disease)

insert human gene

Infect the human with the engineered

virus

The virus is the delivery system

46

Gene Therapy ADA 1990

Genetic defect in white blood cells

Severe immune deficiency

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Remove wbc

Engineer in lab

Infuse into patient

Repeat

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Bacteriophage phenotypes

virulent phage - always lytic, cannot

become a prophage

temperate phage - lysogenic

Temperate phage

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Transformation

Naked DNA enters bacterial cell. Brings new

genes

Plasmids are extrachromosomally maintained

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Plasmids are cloning vectors

(ch 8 pg 179)

pUC19 plasmid, a cloning vector

ampr gene

ori

restriction sites

(multiple cloning site)

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Ampr

Ori

araC

GFP

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Transformation in the laboratory

Make cells competent by calcium chloride

42 degree C heat shock facilitates uptake

Both have ori

55E. Coli genome is 4.6 million bp pGlO is 5,371 bp

genome

Bla gene encodes ampicillin

resistance

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Ampicillin antibiotic inhibits cell wall

synthesis and cell lyse

Bla gene encodes beta lactamase which

breaks down ampicillin

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LABORATORY

E. coli bacteria strain K12/HB101

Non pathogenic

Grows well at room T or 37oC

Prototroph grows in Luria broth (LB)

Host for plasmid DNA

Killed by ampicillin (if untransformed)

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Aequorea victoria – source of the GFP gene

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Engineering the plasmid, pGLO

1. Isolate jellyfish DNA

2. Use restriction enzymes to

cut out GFP gene

3. Purify GFP gene

4. Ligate GFP into plasmid

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pGLO plasmid

ori –replication of plasmid

Ampr (bla)- ampicillin resistance

Only transformed bacteria can grow

in presence of amp

GFP gene

Ara C GFP gene expressed in presence of

arabinose sugar

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GFP gene cloned into plants

Arabidopsis thaliana

seedlings

Reporter gene

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C. elegans

GFP a reporter for olfactory receptor gene

expressed when worms sense the odorant,

diacetyl

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M. musculus (mouse)

GFP reporter for MHC

gene

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GFP embryoGFP mother with GFP-

minus embryo

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Every cell has GFP

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Anopheles gambiae cells GFP and the reaper

apoptosis gene

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Hoxc13-GFP fusion protein expression in

nails of embryonic day 14.5 mouse

Brain tumor expressing rfp

GFP and YFP reporter for stem

cells

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Glow fish pets

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The Lac Operon 1961, Jacob and Monod

E. coli and other bacteria

Bacterial Genes

Many genes are constitutively expressed

these are “housekeeping” genes

Other genes are regulated

Can be turned on, or off depending on cell needs

Operon

group of coordinately regulated genes

One promoter for a number of genes

Polycistronic mRNA

1 mRNA molecule has info from multiple

genes

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E. Coli Lac Operon

E. coli cells can convert lactose to glucose and

galactose

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The Lac Operon allows for

coordinate gene expression

Note: 1 mRNA, promoter

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3 STUCTURAL GENES = Z, Y, A

Lac Z gene encodes b-galactosidase enzyme

b-gal

lactose ------------- glucose + galactose

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LacZ gene is only transcribed when lactose

sugar is present

b- gal is an inducible enzyme

(induced by lactose from 5 copies enzyme to 1000s)

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Fig. 19.2 hydrolysis

This only occurs in the presence of

lactose, the inducer

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Proteins ->

DNA ->

promoter = regulates transcription of ZYA

operator = must be unbound for P to be “open”

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REPRESSOR PROTEIN (I)

Encoded by Lac I gene

Binds to operator

Prevents RNA pol from binding to promoter

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Is this operon ON or OFF?

Is lactose PRESENT or ABSENT?

Lac I, P, O, ZYA genes are CIS elements

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INDUCER (LACTOSE SUGAR)

LACTOSE PRESENT

• Lactose enters

• Binds repressor protein (I) causing a

conformational change

• This pulls the repressor off the operator

• RNA polymerase transcribes genes

• Cell metabolizes lactose

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Lactose (the inducer) enters the cell

Binds repressor protein causing a conformational change

•

83

repressor binds to operator

polymerase cannot bind promoter

no transcription of ZYA genes

No lactose:

84

NO LACTOSE

Lac operon animation

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Operon mutants

Mutant Mutant Phenotype

lac I- constitutive expression because…

Oc constitutive expression because …

P- no expression of operon because …

lac Z- ?

87

Operon on, or off in the absence of

lactose? Presence of lactose?

Lac I- (I- P+O+Z+Y+A+)

Lac Oc (I+P+OcZ+Y+A+)

88

Remember, repressor and polymerase are

proteins which are diffusible

These proteins bind DNA

They act in TRANS

The promoter, operator, and ZYA and I are

genes and cannot move

They act in CIS

Gene cloning in bacteria

1. Isolate DNA from organism

2. Cut DNA and vector with restriction enzyme(s) to

produce overhangs (sticky ends)

3. Ligate to form recombinant DNA

4. Transform bacteria with engineered vector

use selectable marker

5. Grow bacteria

6. Isolate protein from bacteria

Jellyfish DNA

GFP gene

Recombinant plasmid

pGLO

cut plasmid

plasmid

Next

transformation

purify GFP

Crack open the E. coli cells -- purify GFP

Bacterial chromosome plasmid

Pure GFP protein

Human Growth Hormone (hGH) cloned into

bacteria (1980s)

hGH is a 191 aa peptide produced by

the pituitary gland

Pre-1980s hGH purified from

cadaver brains

Drawbacks?

Today

hGH has been cloned

26 inches tall

1. Isolate (normal) human DNA

2. Cut hGH gene out with restriction enzymes

….ggattgcgtacgctttgatcgtagtaataggacctagtgtgtacgtaagc

gg……

….ccattcgcatgcgaaagtagcatcattatccaggatctcacatgcattcg

cc……

pure hGH gene gatcgtagtaatagg

agtagcatcattatcc

3. Ligate hGH gene into plasmid vector = recombinant DNA

4. Transform bacteria

5. Grow bacteria - billions of copies overnight

bacteria will express the hGH gene to make hGH

protein

6. Purify hGH, bottle, sell, and inject before puberty

Reported effects include decreased body fat,

increased muscle mass, increased bone

density, increased energy levels, improved

skin tone and texture, and improved immune

system function.

Banned by IOC and NCAA (and others)

Other cloned drugs made by bacteria

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

Vaccines (hepatitis B surface antigen)

Gene library

Genomic library = Collection of clones that

contain the entire genome

Need > 50,000 bacterial clones to hold the

entire human genome?

Getting a gene into bacteria

*Transformation (plasmid)

Infection (phage)

Gene gun

Screening a library fig.

8.11

Each colony contains a

different fragment of DNA

They are unordered!

Need many plates!

cDNA library (coding regions only) = made

from mRNA

Tissue specific library

Chromosome specific library

Caveats

1. Restriction enzymes may cut within genes

2. Need vast numbers of recombinant bacteria

to represent entire genome

DNA libraries – Ch. 8

Genomic library– how many cells are needed

to represent the entire human genome (46

chromosomes, 3 billion nucleotide base

pairs)?

cDNA library – what type of genes would be

represented in a cDNA library of stomach

lining?

Chromosome specific library – how many

chromosome libraries are required to

represent the entire human genome?

Genomic: Need many bacterial transformants to

represent human genome

http://www.accessexcellence.org/RC/VL/GG/ecb/ecb_images/10_23_genomic_library.jpg

Alcohol dehydrogenaseLane 1 RNA marker

Lane 2 total RNA (Liver)

Lane 3 Brain

Lane 4 Cerebellum

Lane 5 Cerebrum

Lane 6 Kidney

Lane 7 Liver

Lane 8 Lung

Lane 9 Spleen

Lane 10 Thymus

Lane 11 Testis

104Northern blot to assay mRNA levels in various tissues

cDNA library:Each tissue type expresses different genes

Chromosome specific library

Lac operon animation

Cloning into plants (GM) Ch. 9

Transgenic plants

Plants that acquire a new genetic trait by

direct introduction of gene

Inject gene into plant embryo (or plasmids

can be used)

How to make a transgenic plant

Isolate non- edible (wild) tomato plant DNA

Cut DNA with restriction enzyme

Cut out the gene that encodes sweetness

(restriction enzyme)

Cut plasmid (Ti)with same enzyme

Ligate to form rDNA

transform other plant embryo tissue

culture

How to make a transgenic tomato

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

The corn root worm

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

A transgenic animal carries a foreign gene

deliberately inserted into its genome.

Transgenic goats Ch. 9

Produce human protein

(drug) in milk

Pharming

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 do we know if kid

is transgenic?

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

Pail of milk with EPO Bottled EPO drug

Transgenic

animal

contains one

foreign gene

Other proteins made in transgenic sheep and goat milk

• 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.

genus Araneus

• 2009: FDA Approves Drug From Transgenic Goat Milk ATryn, human antithrombin protein

• Anti HIV protein

• Anticancer drugs

• Alpha1-antitrypsin for emphysema

Mouse model organism

These mice are

models for human

disease (Alzheimer)

This mouse is

genetically modified

to be diabetic

Knockout mice

Normal gene (in embryo) has been replaced

with non-functional gene

Examples

Cystic Fibrosis (CF) - The Cftr knockout

mouse

Cancer - p53 knockout mouse has a disabled

Trp53 tumor suppressor gene

Glaucoma - The DBA/2J mouse exhibits many

of the symptoms that are often associated with

human glaucoma

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

Medicine

This chicken produces a human antibody in

her eggs

Xenotransplantation

Pigs have similar sized organs to humans

May involve knocking out pig cell surface

antigens

to prevent hyperacute rejections

Fish farming

genetically engineered salmon grow faster (not FDA

approved)

The fish that has been mainly caught in the

nets of criticism is an Atlantic salmon with a

growth hormone gene from Chinook

salmon.

Researchers in Cuba and the UK have

engineered tilapia to grow and put on

weight up to 300% 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

3 types of cloning

1. gene cloning

Recombinant bacteria (as in lab)

Transgenic plants

Transgenic animals

2. reproductive cloning

Yields an organism

Embryo twinning or nuclear transfer

3. therapeutic cloning

nuclear transfer for stem cells to treat

disease

Reproductive cloning

Embryo twinning

1 sperm + 1 egg - 2 embryos (genetically

identical)

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

atiscloning/

Nuclear transfer method - The clone’s

DNA is a genetic copy of the donor

SCNT =

somatic cell

nuclear

transfer

pg. 577

1997 Ian Wilmut

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

3. Place nucleus into enucleate egg

4. Grow for 6 days in lab

5. Implant into surrogate mother

277 embryos -> 1 lamb (Dolly)

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

Somatic cell nuclear transfer

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

Cloning game

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

Why clone animals?

Models for disease

Pharming

Endangered species – ex. Mouflon sheep,

the surrogate mother was a domestic sheep!

Reproduce deceased pet

Help infertile couples

$1395 PetBank provides pet lovers with the option to

clone their exceptional pets. If you don't save your pet's

DNA today, cloning may not be possible tomorrow.

Saving your pet's DNA is simple, safe and lasts forever.

$150 annual storage fee after the first year.

K.C., the first

animal produced

by cloning from a

cell taken from a

carcass, was born

in April 2002.

Yoda and Sue were

cloned from a

Landrace boar in

2002.

"FDA: Food from animal

clones safe to eat"

Associated Press, October

31, 2003

A Boca Raton, Florida, couple paid a California firm $155,000

to clone their beloved Labrador retriever, who died from cancer

a year ago. The clone, a 10-week-old puppy dubbed Lancey,

was hand-delivered to them earlier this week by Lou

Hawthorne, chairman of BioArts International, a biotechnology

company.

.

Problems with reproductive cloning

High failure rate < 3% success rate 2003 first horse cloned (Prometea) 22 embryos, 800

eggs

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.

Dark brown = permissive policy light brown = flexibleYellow = no federal government funding