Chapter 19

BIOTECHNOLOGY

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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
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Many Important Medicines Are Produced by Recombinant Microorganisms

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

PO

lacZ

b-galactosidase

CNBr cleaves the peptide bond after methionine.

ampR

Insulin B chain

Met

Met b-galactosidase B chain B chain Active insulin

Transform into E. coli.

Culture cells.

Purify -galactosidase-. Treat with CNBr. Purify A and insulin fusion proteins. B chains. b-galactosidase Met Met b -galactosidase A chain A chain Refolding and Disulfide bond disulfide bond formation

PO

lacZ

Insulin A chain

Figure 19.1 The use of bacteria to make human insulin

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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
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Microorganisms Can Reduce Environmental Pollutants

The term bioremediation refers to the use of microorganisms to reduce environmental pollutants 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 pollutants include petroleum hydrocarbons, pesticides, herbicides, organic solvents, etc.

In many cases, biotransformation results in biodegradation

The toxic pollutant is degraded into nontoxic metabolites
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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

The elimination of a genes function from an organism like the mouse, for example, can help us understand the normal function of that gene in mammals
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Genes can be introduced from different species. Here, different fluorescent proteins from jellyfish are expressed in zebrafish.

Figure 19.5

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Figure 19.3

The right mouse expresses the human growth hormone gene

Production of Mice That Contain Gene Knockouts or Gene Replacements

We use model genetic organisms to study the effects of mutations in particular genes, which tells us about the function of those genes
Loss-of-function mutations Gain-of-function mutations RNAi

Mice are the model genetic organism most closely related to humans Altering the expression of mouse genes can help us better understand the function of these genes in humans
Knocking out mouse genes may teach us about the nature of particular human diseases, for example
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Production of Mice That Contain Knockouts or Replacements

The process of homologous recombination, where two homologous sequences pair with each other and then DNA is exchanged, is used to create gene KOs or replacements
The cloned gene contains a neomycin-resistant gene (NeoR) 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 The modified target gene is then introduced into mouse embryonic cells which can be grown in the lab
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Figure 19.6
The gene of interest has been cloned. A neomycin resistance gene is inserted into the center of this gene, and a thymidine kinase gene is inserted next to the gene.

TK

NeoR

Gene of interest

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

Embryonic stemc ells

Normal gene TK NeoR

Chromosomal DNA NeoR

Sensitive to gancyclovir

Dies

Resistant to both drugs

Survives

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Figure 19.6

NeoR

Surviving cells are injected into embryonic blastocysts derived from a mouse with white coat color. The injected blastocysts are reimplanted into the uterus of a female mouse.
Blastocyst

Survives

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

Chimeric offspring

Following birth, chimeric mice are identified as those that contain a coat with both dark and white fur. The appropriate crosses are made in order to produce mice that have two copies of the target gene.

Alan Handyside, Wellcome Images

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Gene Knockouts help scientists understand human diseases

Gene knockouts may reveal the function of the gene Mouse models have been useful in understanding cancer, obesity, heart disease, diabetes and many inherited diseases
A strain of mice engineered to carry a mutation analogous to a disease-causing mutation in a human gene is termed a mouse model

Sometimes there is no obvious phenotype, though

Single gene may only make small contribution to overall phenotype Another gene with similar function may compensate (gene redundancy)

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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 cells genome via 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

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+ Cloned gene Normal gene Normal gene Homologous recombination

+ Cloned gene

If the cloned gene was rendered inactive by mutation => gene knockout

Nonhomologous recombination

Cloned gene

Normal Cloned gene gene

(a) Gene replacement

(b) Gene addition

Figure 19.4

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Gene Knockin (gene addition)

A Gene Knockin is a gene addition in which a gene of interest has been added to a particular site (usually noncritical) in a genome.
Nucleus of a mouse embryonic stem cell Gene of interest

Mouse chromosome

Noncritical site

The gene of interest is cloned with flanking pieces of DNA from a noncritical site in the mouse genome. The cloned DNA is introduced into an embryonic stem cell.

The gene of interest inserts into noncritical site by homologous recombination.

Figure 19.7

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Table 19.3

Human hormone gene Using recombinant DNA technology (described in Chapter 18), clone a human hormone gene next to a sheep -lactoglobulin promoter. This promoter is functional only in mammary cells so that the protein product is secreted into the milk. -lactoglobulin promoter

Plasmid vector Inject this DNA into a sheep oocyte. The plasmid DNA will integrate into the chromosomal DNA, resulting in the addition of the human hormone gene into the sheep's genome. Implant the fertilized oocyte into a female sheep, which then gives birth to a transgenic sheep offspring.

Transgenic sheep

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

molecular pharming

Purify the hormone from the milk.

Figure 19.8

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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!
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Donor sheep's mammary cell is extracted and grown in a tissue culture flask. Another sheep's unfertilized egg is extracted, and the nucleus is removed.
Mammary cell Nucleus

Donor sheep

Unfertilized egg The cells are fused together with electrical pulses.

Mammary cell
Egg with nucleus removed The donor nucleus from the mammary cell and the maternal proteins within the enucleated egg initiate development of the egg into an embryo.

Figure 19.9 Protocol for the

successful cloning of sheep

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The embryo is transferred into a surrogate ewe.

Surrogate ewe

Allow pregnancy to proceed.

Figure 19.9 Protocol for the

successful cloning of sheep

A lamb genetically identical to the donor sheep is then born.

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 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, Dollys 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
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 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

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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

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Figure 19.11
Stem cell

Cellular division

Stem cell
Differentiation + Red blood cell Cellular division Stem cell

Differentiation + Red blood cell

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

Fertilized egg (totipotent)

Can produce all cell types in an adult organism Can give rise to an entire organism

Inner cell mass

Embryonic stem cells (ES cells) (pluripotent)

Blastocyst

Can produce almost all cell types in an adult organism However, a single cell has lost the ability to produce an entire organism

Figure 19.12

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Certain types of human cancers called teratocarcinomas arise from pluripotent cells

These cells are called embryonic carcinoma cells (EC cells)

Embryonic germ cells (EG cells) (pluripotent)

Fetus

Can differentiate into several cells types For example, hematopoietic stem cells (HSC cells) of the bone marrow

Many types of adult stem cells (multipotent or unipotent) Can only differentiate into a single cell type For example, primordial germ cells in the testis sperm, only

Figure 19.12

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Teratomas are germ cell cancers

RadioGraphics March 2001 vol. 21 no. 2 475-490

A Multipotent stem cell can give rise to many different cell types

Hematopoietic stem cell

Cell division Hematopoietic stem cell + Hematopoietic progenitor

or Myeloid progenitor Red blood cell Lymphoid progenitor

Megakaryocyte

Dendritic cell Neutrophil

T cell B cell Natural killer cell

Platelets Basophil Osteoclast Monocyte

Eosinophil

Dendritic cell

Macrophage

Figure 19.13 Fates of hematopoietic stem cells

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

Embryonic Stem cells (ES) 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

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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

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Mouse prostate stem cell that has grown into a prostate (attached to a mouse kidney)

Leong et al, Nature 2008

Culture of mouse kidneys in vitro Michos et al., 2008

Recently, scientists have been able to re-program a differentiated cell into an induced pluripotent stem cell (iPS) via gene additions that lead to over-expression of 4 specific transcription factors

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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
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Figure 19.19
Carries a positive charge (cationic)

Liposome DNA carrying the gene of interest

Virus is genetically altered so that it cannot proliferate after entry into host cells
RetrovirusRNA genome contains gene of interest.

RNA genome

Target cell DNA-liposome complex is taken into the target cell by endocytosis. The liposome is degraded within the endosome and the DNA is released into the cytosol. The DNA is imported into the cell nucleus.

Target cell Retrovirus is taken into the target cell via endocytosis. The viral coat is disassembled in the endosome, and two copies of the RNA genome are released into the cytosol. The RNA is reverse transcribed into DNA, which travels into the nucleus.

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Figure 19.19
Endosome Endosome

Reverse transcriptase By recombination, the DNA carrying the gene of interest is integrated into a chromosome of the target cell. By recombination, the viral DNA, carrying the gene of interest, is integrated into a chromosome of the target cell.

Integrated gene

Integrated gene

(a) Nonviral approach

(b) Viral approach

Does not elicit immune response Low efficiency

ADVANTAGE DISADVANTAGE

High efficiency Elicits immune response

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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, and is particularly toxic to B and T cells The destruction of these cells leads to a disease termed severe combined immunodeficiency (SCID), typically fatal at an early age

Treatments included
Bone marrow transplant Purified ADA protein Gene therapy
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Figure 19.21

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Gene Therapy
Results of treatment were not conclusive
ADA gene was expressed in only a small percentage of cells

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

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