Chapter 19 *Lecture Outline Copyright © The McGraw-Hill Companies, Inc. Permission required for...

Chapter 19

*Lecture Outline

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

*See separate FlexArt PowerPoint slides for all figures and tables pre-inserted into PowerPoint

without notes.

INTRODUCTION

• Biotechnology is broadly defined as technologies that involve the use of living organisms, or their products, to benefit humans

• It is not a new topic– It began about 12,000 years ago when humans began to domesticate

animals and plants for the production of food

• Since the 1970s, molecular genetics has provided new, improved ways to make use of organisms to benefit humans– Genetically modified organisms (GMOs) have received genetic

material via recombinant DNA technology– An organism that has integrated recombinant DNA into its genome is

called transgenic

19-2Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

19.1 USES OF MICROORGANISMS IN BIOTECHNOLGY

• Microorganisms are used to benefit humans in various ways– Refer to Table 19.1

• Molecular genetic tools are very important in influencing and improving our use of microorganisms

• Overall, the use of recombinant microorganisms is an area of great research interest and potential– However, there are problems such as safety concerns and negative

public perception

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-3

Many Important Medicines Are Produced by Recombinant Microorganisms

19-5

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Insulin regulates several physiological processes, particularly the uptake of glucose into fat and muscle cells

It is produced by the cells of the pancreas Persons with insulin-dependent diabetes have a defect in

their cells Therefore, they cannot synthesize enough insulin

Sources of insulin included Cows Human cadavers!

But now, patients can use insulin made by recombinant bacteria

Refer to Figure 19.1

19-6

Insulin

P O

P O

lacZ

lacZ

ampR

galactosidase

galactosidase

galactosidase

galactosidase

Met

CNBr cleavesthe peptidebond aftermethionine.

Met

B chain

InsulinB chain

B chain

Treat with CNBr.Purify β-galactosidase-.insulin fusion proteins.

Culture cells.Transform into E. coli. Purify A andB chains.

Active insulin

Disulfide bondRefolding anddisulfide bondformation

Met

A chain

InsulinA chain A chain

Met


Figure 19.1 The use of bacteria to make human insulin19-7

Insulin is a hormone composed of two polypeptide chains, called the A and B chains

Bacterial Species Can Be Used as Biological Control Agents

• Biological control refers to the use of microorganisms or their products to alleviate plant problems– Disease or damage from environmental conditions

• Biological control agents can prevent disease in one of two main ways:– 1. Nonpathogens are used to compete effectively against

pathogens for nutrients or space– 2. Microorganism may produce toxins that inhibit other

microorganisms or insects, but not the plant


• Biological control can also involve the use of microorganisms in the field– A successful example is the use of Agrobacterium

radiobacter to prevent crown gall disease caused by Agrobacterium tumefaciens

– A. radiobacter produces agrocin 84, an antibiotic that kills A. tumefaciens

• A. radiobacter contains genes that confer resistance to agrocin 84

– The genes responsible for agrocin 84 synthesis and resistance are on a plasmid

• Unfortunately, this plasmid can be transferred via conjugation

– Researchers have found A. radiobacter strains with plasmids that have lost the ability to be transferred

• These conjugation-deficient strains are now used commercially worldwide to prevent crown gall disease


• Biological control can also involve the use of microorganisms in the field– Another successful example is the use of Bacillus

thuringiensis toxins. • These ‘Bt’ toxins are lethal to many caterpillars and beetles• Generally harmless to plants and humans• Bt toxins are sold in powder form

– Can be used as foliage spray

– Toxins paralyze insect’s digestive tract

• Will discuss later how Bt is used to produce transgenic plants


The Release of Recombinant Microorganisms into the Environment

• A recombinant microorganism is one whose DNA is altered in vitro and then reintroduced back into it– Mutations and acquisition of naturally-occurring plasmids

do not create recombinant strains!!!

• This is an important distinction from the perspective of governmental regulation and public perception– Each year new strains of nonrecombinant microorganisms

are analyzed in field tests for biological control of plants– However, the release of recombinant microorganisms is

controversial


• As an example, let’s consider the first field test of a recombinant bacterium– It was carried out by Steven Lindow and colleagues– It involved the use of genetically-engineered Pseudomonas

syringae to control frost damage• Ice+ strains produce proteins that promote ice nucleation• Ice– strains do not make ice-nucleation proteins • When applied to the surface of plants, an Ice– strain can compete

with, and thereby reduce, the proliferation of Ice+ bacteria

– Lindow sought approval for field tests of an Ice–

recombinant strain in Tulelake, California• These tests were delayed several years by a lawsuit• When the tests were finally approved in 1987, vandals struck!• Nevertheless, the results of the field experiment did show that Ice–

bacteria did protect potato plants from frost damage (Figure 19.2)• However, the product was never commercialized


Microorganisms Can Reduce Environmental Pollutants

• The term bioremediation refers to the use of microorganisms to reduce environmental pollutants– During bioremediation, enzymes produced by a microorganism

transform the structure of the toxic pollutant– This event is called biotransformation

• In many cases, biotransformation results in biodegradation– The toxic pollutant is degraded into nontoxic metabolites

• Biotransformation without biodegradation can occur– In these cases, the pollutant is rendered less toxic by

• Oxidation or reduction reactions• Polymerization reactions


• Since the early 1900s, microorganisms have been used in the treatment and degradation of sewage– More recently, the field of bioremediation has expanded

into the treatment of hazardous and refractory wastes• These wastes are associated with chemical and industrial activity

– These pollutants include petroleum hydrocarbons, pesticides, herbicides, organic solvents, etc.

• In 1980, the U.S. Supreme Court ruled that a live, recombinant microorganism is patentable– As a “manufacture or composition of matter” – The first patented organism was an “oil-eating” bacterium

• But alas, it has not been a commercial success – It can metabolize only a few of the 3,000 or so toxic compounds in

crude oil


19.2 GENETICALLY MODIFIED ANIMALS

• The production of transgenic animals is a relatively new, exciting area of biotechnology– It holds great promise for innovations in

biotechnology• Of course, this is predicated on public acceptance!

• Figure 19.3 shows a transgenic mouse that expresses the human growth hormone gene


Figure 19.3 19-16

Gene Addition versus Gene Replacement

• Cloned genes can be introduced into plant and animal cells– However, the gene will not be inherited stably if it does

not become integrated into the host cell’s genome• This integration occurs by recombination

• The introduction of a cloned gene into a cell can lead to one of two outcomes– Gene replacement (can lead to gene knockout if a

defective copy replaces a good copy.)– Gene addition

– Refer to Figure 19.4Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-17

+ Cloned gene

Homologousrecombination

Normalgene

Clonedgene

(a) Gene replacement

+ Cloned gene

Nonhomologousrecombination

Normalgene

Clonedgene

Normalgene

(b) Gene addition


19-18Figure 19.4


If the cloned gene was rendered

inactive by mutation => gene

knockout

19-19Figure 19.5


Genes can be introduced from different species. Here, different fluorescentproteins from jellyfish are expressed in zebrafish.

Production of Mice That Contain Gene Replacements

• In bacteria and yeast, gene replacement is the common outcome– These have relatively small genomes, so homologous

recombination occurs at a relatively high rate

• In complex eukaryotes, gene addition is the norm– These have very large genomes, so homologous

recombination is rare• Only 0.1% of the time


• To produce mice with gene replacements, molecular biologists have resorted to trickery– Cells in which homologous recombination has occurred are

preferentially selected

• This approach is shown in Figure 19.6– The cloned gene is altered using two selectable markers

• A neomycin-resistant gene (NeoR) is inserted into the center of the coding sequence of the target gene

• A thymidine-kinase gene (TK) is inserted adjacent (not within) the target gene

– TK renders cells sensitive to killing by a drug called gancyclovir

– The modified target gene is then introduced into mouse embryonic cells which can be grown in the lab


NeoRTK

Geneof

interest

Normalgene

NeoR

ChromosomalDNA

TK

Dies

NeoR

Survives

This cloned DNA is then introduced into embryonicstem cells. In this case, the cells were derived froma mouse with dark fur color. The cells are grown inthe presence of neomycin and gancyclovir. Only those cells that contain the NeoR gene but are lacking the TK gene will survive.

The gene of interest has been cloned. Aneomycin resistance gene is inserted intothe center of this gene, and a thymidinekinase gene is inserted next to the gene.

– Same gene as normal,chromosomal gene exceptit has NeoR inserted into it.

Nonhomologousrecombination

Embryonic stemc ells

Homologous

recombination

19-22

Figure 19.6

Sensitive to gancyclovir

Resistant to both drugs


Blastocyst

Chimeric offspring

Surviving cells are injected into embryonicblastocysts derived from a mouse with white coatcolor. The injected blastocysts are reimplanted intothe uterus of a female mouse.

© Alan Handyside, Wellcome Images

NeoR

Survives

19-23

Figure 19.6

A chimera is an organism that contains cells from two different individuals


Following birth, chimeric mice areidentified as those that contain acoat with both dark and white fur.The appropriate crosses are madein order to produce mice that havetwo copies of the target gene.


19-24

Gene Knockouts help scientists understand human diseases

• Gene knockouts may reveal the function of the gene• Sometimes there is no obvious phenotype

– Single gene may only make small contribution to overall phenotype– Another gene with similar function may compensate

• This is called gene redundancy

• Knocking out mouse genes may teach us about human disease– Mice and humans share many genes

• Seeing characteristics of knockout mice may lead to treatments of human diseases.

– Mouse models have been useful in understanding cancer, obesity, heart disease, diabetes and many inherited diseases.


Gene KnockinA Gene Knockin is a gene addition in which a gene of interest has been

added to a particular site (usually noncritical) in a genome.

19-25

Figure 19.7

The gene of interest iscloned with flankingpieces of DNAfrom a noncriticalsite in the mousegenome. The clonedDNA is introducedinto an embryonicstem cell.

The gene ofinterest inserts intononcritical site byhomologous recombination.

Nucleus of a mouseembryonic stem cell

Mousechromosome

Gene of interest

Noncriticalsite



Transgenic Livestock• Transgenic species of livestock are being developed

– Includes fish, sheep, pigs, goats and cattle as well as others

• May include production of medicines in the milk of these animals– Sometimes called molecular pharming– See Figure 19.8 and Table 19.3

19-26


Figure 19.8

Table 19.3

Purify the hormone from the milk.

Obtain milk from female transgenic sheep.The milk contains a human hormone.

Inject this DNA into a sheep oocyte. Theplasmid DNA will integrate into thechromosomal DNA, resulting in the additionof the human hormone gene into the sheep'sgenome.

Implant the fertilized oocyte into a femalesheep, which then gives birth to a transgenicsheep offspring.

Using recombinant DNA technology (describedin Chapter 18), clone a human hormone genenext to a sheep β-lactoglobulin promoter. Thispromoter is functional only in mammary cellsso that the protein product is secreted into themilk.

Humanhormonegene

Plasmidvector

Transgenicsheep

β-lactoglobulinpromoter


19.3 REPRODUCTIVE CLONING

• Reproductive cloning refers to methods that produce two or more genetically identical individuals– Identical twins are genetic clones from one fertilized egg

• Cloning is an easier undertaking in plants– Plants can be cloned from somatic cells

• For several decades scientists believed that mammalian somatic cells were unsuitable for cloning– But in 1997, Ian Wilmut and his colleagues at the Roslin

Institute created Dolly!


Donor sheep's mammary cellis extracted and grown in atissue culture flask. Anothersheep's unfertilized egg isextracted, and the nucleus isremoved.

The donor nucleus fromthe mammary cell andthe maternal proteinswithin the enucleated egginitiate development ofthe egg into an embryo.

Mammarycell

Donor sheepUnfertilized egg

Nucleus

Mammary cell

The cells are fusedtogether with electricalpulses.

Egg with nucleusremoved

19-29

Figure 19.9 Protocol for the successful cloning of sheep



19-30

Figure 19.9 Protocol for the successful cloning of sheep


The embryo istransferred into asurrogate ewe.

Allow pregnancyto proceed.

Surrogateewe

A lamb geneticallyidentical to the donorsheep is then born.


• Evidence suggested that Dolly may have been “genetically older” than her actual age would have indicated– At 3 years old, the length of the telomeres in her somatic

cells were consistent with a sheep that is 9 or 10 years old

• The sheep that donated the somatic cell that produced Dolly was 6 years old– Thus, Dolly’s shorter telomeres were likely a result of the

shortening of telomeres in the donor sheep

• In 2003, 6-year old Dolly was euthanized after an examination showed progressive lung disease– Her death raised concerns that the techniques used to

produce Dolly could have caused premature aging– Microarray studies in cloned mice showed as much as 4%

of genes were not expressed normally


• In recent years, cloning from somatic cells has been achieved in several mammalian species– Sheep, cows, mice, goats and pigs– Unlike the case with Dolly, telomeres in cloned mice and

cattle appear to be the correct length!– However, other studies have shown other genetic flaws

• With regard to livestock, farmers can use somatic cells from their best individuals to create genetically homogeneous herds– This may be advantageous with regard to agricultural yield– However, such a herd may be more susceptible to rare

diseases


• People have become greatly concerned with the possibility of human cloning– To some, it is morally wrong and threatens the basic fabric

of parenthood and family– To others, it offers a new avenue of reproduction

• For infertile couples who might want a genetically related child

• In the public sector, the sentiment toward human cloning has been generally negative– Indeed, many countries have issued an all-out ban – While others permit limited research in the area

• In the future, our society will have to wrestle with the legal and ethical aspects of cloning


Stem Cells • Stem cells supply the cells that construct our bodies

from a fertilized egg– In the adult, stem cells also replenish damaged cells

• Stem cells have two common characteristics– 1. They have the capacity to divide – 2. They have the capacity to differentiate into one or more

specialized cell types• totipotent cells, like fertilized eggs can give rise to all cell types• pluripotent cells can differentiate into almost every cell, but can’t

give rise to an entire, intact individual• multipotent cells can differentiate into several cell types• unipotent cells can only differentiate into one cell type

– Refer to Figures 19.11 and 19.12


19-35Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 19.11

• When a stem cell divides, one may remain undifferentiated, while the other can differentiate into a specialized cell type– Thus the population of stem cells remains constant


Cellular division

Cellular division

Stem cell

Red blood cell

Red blood cell

+

+

Stem cell

Stem cell

Differentiation

Differentiation

Blastocyst

Inner cellmass

Embryonic stem cells (ES cells)(pluripotent)

Fertilized egg(totipotent)


19-36

• In mammals, stem cells are commonly categorized based on their developmental stage and their ability to differentiate

Figure 19.12Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Can produce all cell types in an adult organism

Can give rise to an entire organism

Can produce almost all cell types in an adult organism

However, a single cell

has lost the ability to produce an entire organism

Found in the early mammalian embryo

Fetus

Embryonic germ cells (EG cells)(pluripotent)

Many types of adultstem cells(multipotent orunipotent)

19-37Figure 19.12


Certain types of human cancers called teratocarcinomas arise

from pluripotent cells

These cells are called embryonic carcinoma cells

(EC cells)

Can only differentiate into a single cell type

For example, primordial germ cells in the testis

sperm, only

Can differentiate into several cells types

For example, hematopoietic stem cells (HSC cells) of the bone

marrow


19-38

Figure 19.13 Fates of hematopoietic stem cells


A Multipotent stem cell can give rise tomany different cell types

Hematopoieticstem cell

Hematopoieticstem cell

+

or

Myeloidprogenitor

Red blood cell

Basophil

Platelets

Megakaryocyte

Osteoclast

Monocyte

Macrophage

Eosinophil

Neutrophil

Dendriticcell

Lymphoidprogenitor

Hematopoieticprogenitor

Cell division

T cell

B cell Naturalkiller cell

Dendriticcell


Stem Cells

• Interest in stem cells centers around two main areas– 1. They may help us understand the basic genetic

mechanisms that underlie the process of development – 2. They offer the potential to treat human diseases or

injuries that cause cell and tissue damage• This application has already become a reality

– For example, bone marrow transplants are used to treat patients with certain forms of cancer

– As shown in Table 19.4, embryonic stem cells could potentially be used to treat a wide variety of diseases


Stem Cells

• Adult stem cells are rare– 1 cell in 10,000 in the bone marrow is a stem cell

• Embryonic Stem cells (ES) and Embryonic Germ cells (EG) can be grown in the laboratory– Are easily identified– Provide greatest potential for transplantation therapy– Most ES cells are derived from unused embryos from

in vitro fertilization– Most EG cells are derived from aborted fetuses– This creates an ethical dilemma in using these cells


19.4 Genetically Modified Plants• Selective breeding has been used for centuries

– This has produced plants with desirable characteristics • larger, disease resistance, high-quality food

• Genetically engineered crops have been used since mid-1990s– In 2009, roughly 25% of all crops were transgenic– More than 100 million hectares planted with transgenics


A. Tumefaciens Can Be Used to Make Transgenic Plants

• The production of transgenic plants is somewhat easier than transgenic animals– Certain plant cells are totipotent

• An entire organism can be regenerated from a somatic cell

• Agrobacterium tumefaciens is a bacterium that naturally infects plants causing crown gall tumors– Ti plasmid (Tumor-inducing) carried by bacterium– Refer to Figure 19.14



19-44

Tumor-inducing plasmid

Transferred DNA

Figure 19.14Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

During infection, the T DNA within the Ti plasmid istransferred to the plant cell. The T DNA becomesintegrated into the plant cell's DNA. Genes within theT DNA promote uncontrolled plant cell growth.

Plantchromosome

T DNA A. tumefaciens

Ti plasmid

Plant cell

Woundsite

Agrobacterium tumefaciens is found within the soil.A wound on the plant enables the bacterium toinfect the plant cells.

T DNA

T DNA

19-45Figure 19.14


T DNA

T DNA

The growth of the recombinant plant cells producesa crown gall tumor.

(a) The production of a crown gall tumor by A. tumefaciens infection

Crowngalltumor

T DNA

T DNAT DNAT DNA

T DNAT DNA


• The A. tumefaciens T DNA can be used as a vector to introduce cloned genes into plants

• First, the Ti plasmid needs to be modified– The genes that cause tumors are deleted– Selectable marker genes are inserted into the T DNA

• KanR is commonly used

– Unique restriction sites are added for the convenient insertion of any gene

• Figure 19.15 shows the general strategy for producing transgenic plants via T DNA-mediated gene transfer


19-47Figure 19.15



Gene of interest is inserted intothe T DNA of the T-DNA vector.

Gene of interestT-DNA vector

KanR

(conferskanamycinresistance)

T DNASite whererestrictionenzyme cuts

RecombinantT-DNA vector

The recombinant T-DNA vector istransformed into A. tumefaciens.

Plant cells are exposed to A. tumefaciens.The T DNA is transferred and incorporatedinto the plant cell chromosome. Carbenicillinand kanamycin are then added to killA. tumefaciens cells and plant cells thathave not taken up T DNA, respectively.

RecombinantT-DNA vector

A. tumefaciens

Plantcell

The plant cells are transferred to a mediumcontaining growth hormones to regeneratean entire plant.

Inserted T DNA carrying new gene

Plant with cloned gene

19-48Figure 19.15


T DNA integrates into

the genome


• A. tumefaciens does not infect all plant species– Fortunately, other methods are available for introducing

genes into plant cells

– Biolistic gene transfer (i.e., biological ballistics)• The second most common way to produce transgenic plants • A “DNA gun” is used to shoot DNA-coated microprojectiles into the

cells

– Microinjection• Microscopic-sized needles are used to inject DNA into the cells

– Electroporation• An electric current is used to create transient pores in the plasma

membrane through which DNA can enter


Transgenic Plants

• Transgenic plants can be given characteristics that are agriculturally useful– For example, the Monsanto Company has produced plants

highly tolerant of glyphosate• The active ingredient in the herbicide RoundupTM

– Compared to nontransgenics, these plants grow quite well in the presence of glyphosate-containing herbicides

– Plants have been made more disease resistant

• Table 19.5 presents some of the various traits that have been modified in plants


• Transgenic plants have been approved for human consumption– The first example was the Flavr Savr tomato (Figure 19.18)

• This transgenic plant has been given the gene that encodes an antisense RNA

– Complementary to the mRNA for polygalacturonase (an enzyme involved in fruit ripening)

• The antisense RNA binds to the mRNA and prevents translation• In addition, double-stranded RNA is targeted for degradation

– RNAi-mediated silencing (refer to Chapter 15)

– The practical advantage of the Flavr Savr tomato is improved shelf-life

• It does not spoil (overripen) as quickly as traditional tomatoes


• Transgenic plants can produce their own insecticide– Express genes from the naturally occurring bacterium

Bacillus thuringiensis (Bt)– These genes express toxins that are lethal to many

caterpillars and beetles that feed on trees, shrubs and fruit– Bt corn and cotton are very successful (see Figure 19.18)

• There are concerns about the use of Bt – may kill pollinators of native species– may cause proliferation of resistant insects


Production of Bt crops is rising

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-54Figure 19.18

(a) A field of Bt corn

(b) Bt corn and Bt cotton usage since 1996

Per

cen

t o

f ac

res

100

0

20

40

60

80

Year

Bt cotton

Bt corn

1996 97 98 99 2000 01 02 03 04 05 06 07 08


© Bill Barksdale/AGStockUSA

19.5 HUMAN GENE THERAPY• Gene therapy is the introduction of cloned genes

into living cells in an attempt to cure disease• Research efforts in gene therapy are aimed at

– Alleviating inherited diseases– Treating diseases such as cancer and heart disease– Combating infectious diseases such as AIDS

• Human gene therapy is still at an early stage of development– Success has been limited– Nevertheless, some of the initial results are promising

and future prospects abound– Refer to Table 19.6


Gene Therapy Involves the Introduction of Cloned Genes into Human Cells

• Two transfer methods are used

– 1. Nonviral approach• Refer to Figure 19.19a• Liposome technique most common

– 2. Viral approach• Refer to Figure 19.19b• Most common are retroviruses, adenoviruses and parvoviruses


19-58

Figure 19.19

Carries a positive charge (cationic)

Virus is genetically altered so that it cannot

proliferate after entry into host cells



Retrovirus-RNA genome containsgene of interest.

Liposome

DNA carrying thegene of interest

Target cell Target cell

The liposome is degradedwithin the endosome and theDNA is released into thecytosol.

DNA-liposome complex istaken into the target cell byendocytosis.

The DNA is imported into thecell nucleus.

Retrovirus is taken into thetarget cell via endocytosis.

The RNA is reversetranscribed into DNA, whichtravels into the nucleus.

RNAgenome

The viral coat is disassembledin the endosome, and twocopies of the RNA genome arereleased into the cytosol.

19-59

Figure 19.19

Does not elicit immune response

Elicits immune responseLow efficiency

High efficiencyADVANTAGE

DISADVANTAGE


(a) Nonviral approach (b) Viral approach

Endosome Endosome

Reversetranscriptase

Integratedgene Integrated

gene

By recombination, the DNAcarrying the gene of interest isintegrated into a chromosomeof the target cell.

By recombination, the viralDNA, carrying the gene ofinterest, is integrated into achromosome of the target cell.


Experiment 19A: The First Human Gene Therapy

• Adenosine deaminase (ADA) is an enzyme involved in purine metabolism– If both copies of the gene are defective, deoxyadenosine

will accumulate within the cells of the individual– Deoxyadenosine is particularly toxic to B and T cells– The destruction of these cells leads to a disease termed

severe combined immunodeficiency (SCID)– If left untreated, SCID is typically fatal at an early age


• Three approaches can be used to treat adenosine deaminase (ADA) deficiency – 1. A bone marrow transplant from a compatible donor– 2. Purified ADA coupled to polyethylene glycol (PEG)– 3. Gene therapy

• On September 14, 1990 the first human gene therapy was approved for a girl with ADA deficiency– Prior to the clinical trial, the normal ADA gene had been

cloned into a retrovirus that can infect lymphocytes– The general aim of the therapy was to remove

lymphocytes from a patient with SCID, culture the cells in the laboratory, introduce the normal ADA gene into the cells and reintroduce the cells into the patient



The Hypothesis

Infecting lymphocytes with a retrovirus containing the normal ADA gene will correct the inherited deficiency of the mutant ADA gene in patients with ADA deficiency.


Testing the Hypothesis

Refer to Figure 19.20

19-63Figure 19.20

This is called an ex vivo approach

The genetic manipulations occur outside the body,

yet the products are reintroduced into the body



Experimental level Conceptual level

2. Culture the cells in a laboratory.

1. Remove ADA-deficient lymphocytes from the patient with severe combined immunodeficiency disease (SCID).

Lymphocytes

Lymphocyte

Mutant ADA gene

X

19-64Figure 19.20


Retroviral inserted DNA

3. Infect the cells with a retrovirus that contains the normal ADA gene. Retroviruses insert their DNA into the host cell chromosome as part of their reproductive cycle.

4. Infuse the ADA-gene-corrected lymphocytes back into the SCID patient.

Retrovirus withADA gene LymphocyteRetrovirus

Normal ADA gene

X


AD

A f

un

cti

on

fro

mg

en

e t

her

ap

y(n

mo

les

of

de

am

ina

ted

de

ox

yad

en

osi

ne/

10

8 c

ells

) 20

0

5

10

15

Protocol day

0 365 1460730 1095


19-65Figure 19.20


The Data DNA was

transferred into living cells

Interpreting the Data

• Results of treatment were not conclusive– ADA gene was expressed in only a small

percentage of cells– Drug treatment still needed

• In another test of c cytokine receptor gene therapy, patients developed leukemia– caused by integration of the retroviral vector

used to treat the disease next to a particular gene


Aerosol Sprays May Be Used to Treat Cystic Fibrosis

• Cystic fibrosis (CF) is one of the most common recessive inherited disorders– Affects about 1 of 3,000 babies of northern European

descent– It is caused by a defect in a gene that encodes an ion

transport protein– This leads to an abnormality in salt and water balance– This, in turn, leads to accumulation of mucus in the lungs– The result is chronic lung infections which prove fatal


• CF has been the subject of much gene therapy research– To implement CF gene therapy, the normal CF gene must

be delivered to lung cells• The ex vivo approach used in ADA gene therapy is not possible

• CF gene therapy has involved the use of an aerosol spray – In one approach, the normal CF gene is delivered in an

adenovirus– In another, the gene is delivered via liposomes– When inhaled by the patient via an aerosol spray, the lung

epithelial cells take up liposomes and adenoviruses– Still at an early stage of development– Eventually may become an effective method of treatment


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