Page 15 - Genetics_From_Genes_to_Genomes_6th_FULL_Part1

PART VI Using Genetics
18
chapter
Manipulating the
Genomes of
Eukaryotes
Guided Tour
622 Chapter 18 Manipulating the Genomes of Eukaryotes

Figure 18.5 Transgenic plants produced using a T-DNA makes crown galls to introduce foreign DNA into plants.
Integrating Genetic Concepts
UNTIL RECENTLY, CHILDREN born with poor vision due to Naturally occurring enzymatic processes, whether those
plasmid vector. Researchers infect plants with Agrobacterium
a genetic disease called Leber congenital amaurosis used for DNA repair or for mobilizing transposons or
tumefaciens bacteria containing two plasmid constructs. A T-DNA
Genetics: From Genes to Genomes takes an integrated approach in its presentation of genetics, thereby giving students a
(LCA) were destined to become completely blind by early gene that confers T-DNA, are thus the basis for integrating foreign DNA into
plasmid contains a transgene (Gene) and marker
strong command of genetics as it is practiced today by academic and corporate researchers. Principles are related through-
adulthood. Now, for many of these children, the success host chromosomes.
resistance to an herbicide, both within the T-DNA ends LB and RB. A
out the text in examples, essays, case histories, and connections sections to make sure students fully understand the rela-
helper plasmid contains the vir genes, required for T-DNA transfer to
of gene therapy trials provides hope not only for a halt A statue in front of the Institute of Cytology and Genetics in
a plant cell. Upon infection,
tionships between topics. the recombinant T-DNA integrates into
to the retinal degeneration characteristic of the disease, Novosibirsk, Russia pays homage to the laboratory mouse.
the host plant genome in both somatic cells and eggs. Investigators
but even for restoration of normal sight. © Michael Goldberg, Cornell University, Ithaca, NY
essential concepts
select for single cells or seeds with a transgene insertion by growing
One form of LCA is caused by homozygosity for a then grow the
cells or seeds in the presence of herbicide. They
recessive loss-of-function allele of a gene called RPE65. plant. • Transgenic mice are produced by injecting foreign DNA
selected cell or seedling into a whole transgenic
Chapter Outline
This gene encodes a protein found in the retinal pigment chapter outline into a pronucleus of a fertilized egg.
r
herbicide
Every chapter opens with a brief outline
vir genes
epithelium (a cell layer just beneath the retina) that is cru- RB • Transformation of Drosophila relies on the construction of
Gene
of the chapter contents.LB
cial for the function of photoreceptors. The RPE65 enzyme • 18.1 Creating Transgenic Organisms
transgenes inserted into P element transposon vectors.
functions in the visual cycle—the process by which the • 18.2 Uses of Transgenic Organisms
• Researchers make transgenic plants by infecting plant
retina detects light. LCA patients lose sensitivity to light, • 18.3 Targeted Mutagenesis
cells with Agrobacterium containing a Ti (tumor-inducing)
which eventually results in a reduction in the amount of • 18.4 Human Gene Therapy
plasmid engineered to contain the transgene.
brain cortex devoted to visual processing (Fig. 18.1). 18.3 Targeted Mutagenesis 627

Gene therapy is the manipulation of genes—adding • These methods of creating transgenic organisms result in
Helper plasmid
T-DNA plasmid
DNA to the genome or altering the DNA of a gene—in the integration of transgenes at random locations in the
host genome.
order to cure a disease. The experimental gene therapy strategy for this form of LCA
Transform Agrobacterium
with plasmids and spray
helping scientists to understand the disorder and to develop
A researcher needs only to know the DNA sequence of a
was simple: Scientists delivered normal copies of the RPE65 gene to the retinal pig-
transformed bacteria on plants
more effective therapies.
gene in order to alter it; now that the genome sequences of
ment epithelium cells of patients, simply by injecting DNA packaged in viral particles
Plant cell
Agrobacterium tumefaciens
through the eye into these cells. Since the first results of RPE65 gene therapy clinical
Experiments with primates raise substantial ethical
all model organisms normally used in the laboratory have
18.2 Uses of Transgenic Organisms
concerns for many people, so the future of primate models
trials were reported in 2008, more than 30 patients have undergone the procedure, and
been determined, any gene in these species can be mutated
Recombinant
almost all of them have had their vision restored at least in part; several are no longer
for human genetic diseases is unclear. As of this writing in
at will.
considered legally blind. T-DNA learning objectives
Learning Objectives
2016, the United States National Institutes of Health is in
We focus here mostly on methods to alter specific
transferred to
In this chapter, you will learn about two general strategies for altering genomes:
plant cellasive
the process of phasing out most, though not all, inv
genes in mice, which are the animal of choice for many
Learning Objectives appear before each section, and are care-
research on primate species.
creation of transgenic organisms and targeted mutagenesis. Development of these ex- 1. Describe how transgenes can clarify which gene
studies relevant to human biology. However, at the end of
fully written to clearly outline expectations.
citing technologies has relied on knowledge of the natural processes by which DNA causes a mutant phenotype.
this section we describe an exciting new technique just
Vir proteins
can move within a genome, can be transferred between individuals and between spe-
coming into widespread use that is applicable to many dif-
2. Summarize the use of transgene reporter constructs in
Genomic DNA
r
cies, and can be protected from alteration or degradation. The overarching theme of gene expression studies.
Gene
herbicide
ferent species.
Grow embryos from
essential concepts Recombinant
618 single cells or seeds; add T-DNA 3. Discuss examples of how transgenic organisms serve
integrates into
herbicide to select for
• A wild-type transgene can be inserted into an embryo to produce proteins needed for human health.
plant genome
transformants
homozygous for a recessive mutant allele. If the normal Knockout Mice Have Loss-of-Function
4. List examples of GM organisms and discuss the pros
phenotype is restored, then the transgene identifies the Mutations in Specific Genes
and cons of their production.
gene that was mutated. 5. Explain the use of transgenic animals to model
Transformed plant
gain-of-function genetic diseases in humans.
har00909_ch18_618-645.indd 618 • The creation of reporter constructs allows easy detection Homologous recombination provides a way for DNA se-
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of when and in which tissues a gene is turned on or quences to zero in on specific regions of a genome. In
turned off in eukaryotes. fact, in Chapter 14 you have seen already that gene trans-
• Transgenic organisms produce medically important fer by means of homologous recombination can make
Our ability to generate transgenic organisms has had a
mutations in specific bacterial genes—a process called
Researchers transform A. tumefaciens with two different major impact on biological research and is also increasingly
human proteins including insulin, blood clotting factors,
gene targeting (recall Fig. 14.31). In gene targeting, sci-
and erythropoietin; transgenic crop plants can potentially
plasmids (Fig. 18.5). One is a helper plasmid that contains important for several aspects of daily life. Studies with
make ingestible vaccines.
entists mutagenize a specific gene in vitro, and then in-
the vir genes but no border sequences. The other plasmid is Essential Concepts
transgenic model organisms enable researchers to under-
troduce the mutant DNA into bacterial cells. Homologous
the T-DNA vector engineered to contain the gene to be trans- stand better the functions of particular genes and their regu-
• GM soybeans are resistant to the weed killer glyphosate.
recombination then replaces the normal copy of the gene
Many crops, such as corn, soybean, canola, and cotton
ferred and a marker gene (often a gene that confers resistance After each section, the most relevant points of content are
lation and to model certain human diseases in animals. In
in the bacterial genome with the mutant copy. Although
have been genetically modified to express Bt protein
to an herbicide), both located between the LB and RB now provided in concise, bulleted statements to reinforce
addition, scientists have engineered transgenic plants and
which discourages insect predation.
homologous recombination events are rare, investigators
sequences. To start the infection, investigators spray the crucial concepts and learning objectives for students.
animals to produce drugs and (more controversially) better
can grow large numbers of bacteria easily and then iden -
• Adding a transgene that carries a disease-causing, gain-
transformed A. tumefaciens onto whole plants or plant cells. agricultural products, and even glowing pets (Fig. 18.6).
of-function allele to a nonhuman animal model allows
They next grow individual infected plant cells in culture or tify rare cells containing targeted mutations by selecting
researchers to observe disease progression and to test
seeds in soil to generate embryonic plants, and they select for a drug resistance marker present within the trans -
possible therapeutic interventions.
ferred DNA. Gene targeting in single-celled eukaryotes
embryos or seedlings transformed with the recombinant Transgenes Assign Genes to Phenotypes
T-DNA by adding herbicide to the growth medium (Fig. 18.5). such as the yeast S. cerevisiae by the same method is also
quite routine.
xiv These examples of methods used for constructing In many genetic investigations, the available information
Mouse geneticists use mouse embryonic stem cells
transgenic organisms show how scientists can take advan- may not allow scientists to pinpoint the gene responsible for
(ES cells) to surmount two main obstacles for gene target-
18.3 Targeted Mutagenesis
tage of natural processes to alter genomes. Researchers in a particular phenotype. The construction of transgenic
ing in multicellular organisms. First, for a chromosome
essence have “hijacked” the process by which A. tumefaciens organisms often allows investigators to resolve ambiguities.
containing a targeted gene to be transmitted to progeny,
learning objectives gene targeting has to occur in germ-line cells. Second,
given the low efficiency of homologous recombination,
1. Describe how ES cells are used to generate knockout investigators need to screen through a large number of
mice. germ-line cells to obtain one with the desired mutation.
2. Explain why an investigator might want to create a Mouse ES cells grow in a culture dish, so just as is done
conditional knockout mouse.
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3. Discuss how scientists employ a bacteriophage containing a targeted mutation. A crucial aspect of this
site-specific recombination system to generate procedure is that the ES cells with targeted chromosomes
knockin mice. can be moved from a cell culture dish to a developing
4. Describe CRISPR/Cas9 and how it is used to modify embryo, where they can contribute to all different cell
genomes. types, including germ-line cells.
In the previous section, you saw that genes can be trans- Gene targeting in ES cells
ferred easily into random locations in the genomes of many to generate knockout mice
animals and plants. Here we will explore more advanced Mouse ES cells are undifferentiated cells derived from
technology that enables scientists to change specific genes the inner cell mass of early-stage embryos called blasto-
in virtually any way desired—that is, targeted mutagenesis. cysts (Fig. 18.9). These ES cells are not yet committed to
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