CHAPTER 18: Manipulating the Genomes of Eukaryotes
steps for producing transgenic plants via T-DNA mediated gene transfer
1. A. tumefaciens are transformed w. a T-DNA vector containing a gene of interest and a helper plasmid 2. plant cells are sprayed with the transformed A. tumefaciens 3. individual infected plant cells are grown in culture to generate embryonic plants 4. embryos containing recombinant T-DNA are selected w. herbicide
steps of creating a conditional knockout mouse
1. create a gene targeting construct containing two introns and an exon of the gene to be conditionally knocked out 2. generate ES cells in which sequences in the targeting construct replace the corresponding DNA sequences in the wild-type gene in the mouse genome 3. remove the drug resistance gene with Cre 4. inject ES cells that are heterozygous for the floxed gene into host blastocysts and generate heterozygous mice 5. perform crosses to generate a mouse that is homozygous for the floaxed allel, and also carries a transgene for Cre
Process for creating a knockout mouse
1. the gene of interest is mutagenized by the insertion of a drug resistance marker 2. the DNA constructs are added to the culture medium of embryonic stem cells 3. cells that have incorporated the DNA construct into their genome are selected with drugs 4. PCR is used to ID cell lines whose chromosomes acquired the DNA construct through homologous recombination 5. cells are injected into blastocysts, which are implanted in surrogate mothers 6. Chimeric mice are obtained and can be mated to produce mice with two copies of the inactivated gene
steps for generating transgenic flies with P element vectors
1. the transgene of interest and a marker gene are ligated into a P element vector within the P element inverted repeats 2. the plasmid containing the transgene and a "helper" plasmid are injected into Drosophila embryos at an early stage of development 3. the transposase protein produced by the helper plasmid cuts the P element out of the other plasmid 4. the P element is pasted into a random site in a Drosophila host chromsome 5. the injected embryos mature into adults and are mated and transgenic progeny are selected
sequences contained in the sgRNA used in the genetically engineered CRISPR/Cas9 system
A 20 bp seqeunce that, at its 5' end, is complementary to the genomix target that will be altered a sequence that, at its 3' end, binds Cas9
CRISPR (clustered regularly interspaced short palindromic repeats.)
A region in many bacterial genomes that confers immunity to viral infection. The CRISPR mechanism has been exploited by biotechnologists for genome editing of higher organisms.
Cas proteins
CRISPR-associated proteins. These are bacterial endonucleases integral to the CRISPR immunity system. Cas proteins can cleave viral DNA, leading to its degradation.
to generate a knockin mutant using the CRIPSR/Cas9 sys you would insert in in a cell....
Cas9/sgRNA and a DNA molecule corresponding to the DNA flanking the break
to make transgenic eukaryotes, _____ must be introduced into one or more cells and then it must be replicated or maintiained as the cell divides. additionally, for rhw transgene to be propagated between generations, cells containing the transgene must have the ability to develop into _______-
DNA; gametes
P element transformation is a technique that can be used with what model organism to study gene fxn.
Drosophila
genes used in reporter constructs to detect gene expression
GFP and lacZ
the first transgenic primate model for a human neurological disorder was for ____________ disease
Huntington
Cas9 (CRISPR-associated protein)
RNA-guided DNA endonuclease enzyme associated with the CRISPR, produces single strand breaks in DNA
P element
a DNA transposable element in Drosophila that has been used as a tool for insertional mutagenesis and for germ-line transformation
therapeutic gene
a cloned gene introduced into patient's somatic cells whose product is meant to cure a disease.
marker gene
a gene inserted into a genome along with a transgene whose expression indicates successful incorporation of the transgene.
adeno-associated viral vectors (AAV vectors)
a gene therapy vehicle whose recombinant single-stranded DNA genome contains a therapeutic gene that does not integrate into patient chromosomes.
CRISPR/Cas9 system
a genetically engineered version of the immunity system of Streptococcus pyogenes that is used for genome editing. An investigator-designed sgRNA brings Cas9 endonuclease to a target site in the genome. Cas9 makes double-stranded breaks in the DNA that, through DNA repair, can result in knockouts or knockins.
pronuclear injection
a method for generating transgenic mammals in which DNA is injected into a pronucleus after fertilization.
Agrobacterium-mediated T-DNA transfer
a method for generating transgenic plants where bacteria containing a recombinant plasmid infects plants and the part of the plasmid containing the transgene integrates into the plant genome.
in gene therapy, a therapeutic gene is introduced into....
a patient's somatic cells
ex vivo gene therapy
a procedure where a patient's somatic cells are removed, a therapeutic gene is delivered to the cells in culture, and the cells are reintroduced into the patient.
strategies that are used for choosing a thereapeutic gene
adding a wild-type copy of a gene whose fxn is lacking adding a gene that inactivates a disease gene that is overexpressed adding a gene that might prevent proliferation of tumor cells
chimera
an embryo or animal composed of cells from two or more different organisms.
vaccines
antigens of a disease-causing agent that stimulate an immune response to that particular foreign substance.
first human protein drug produced in the milk of a transgenic animal to be approved by the FDA was...
antithrombin III
transgene
any piece of foreign DNA that researchers have inserted into the genome of an organism.
Cre/loxP recombination system is derived from...
bacteriophage P1 genome
A Bt plant contains genes from a....
bacterium
in vivo gene therapy is used if doctors...
cannot remove cells from the patient's body can access the tissue easily
an organism that contains cells from two different individuals is called a(n)....
chimera
toward the end of the process of creating a knockout mouse, scientists cross ________ mice to wild-type mice to generate mice that have the knockout allele in all cells of their bodies
chimeric
somatic cell nuclear transfer is a technique used in...
cloning
in ________ knockout mice, gene excision is limited to a specific time or tissue because the mouse contains a transgene that expresses Cre at specific time or in a specific tissue
conditional
conditional knockout mice
conditional knockouts transgenic mice that have a floxed gene; the gene can be made nonfunctional through partial deletion in specific tissues.
retroviral and adeno-associated viral vectors used for gene therapy....
contain viral packaging sequences and lack almost the entire viral genome
two general strategies for altering genomes
creation of transgenic organisms and targeted mutagenesis
embryonic stem cells (ES cells)
cultured embryonic cells that continue to divide without differentiating and are capable of becoming any cell type.
knockin mice are used for the study of
dawrfism
in vivo gene therapy
delivery of a therapeutic gene directly to somatic cells.
totipotent
describes a cell state during early embryonic development in which the cells have not yet differentiated and retain the ability to produce every type of cell found in the developing embryo and adult animal.
floxed
describes a gene in a transgenic organism in which an exon is flanked by loxP sites; the gene can be knocked out conditionally in cells that express Cre recombinase.
gene editing
describes a variety of technologies, including CRISPR/Cas9, that allows the creation of knockout and knockin animals without the use of ES cells.
for gene targeting mice, _________ _______ cells are used, as they can be easily screened and cane be added back to a developing embryo
embryonic stem
factors or events involved in successfully producing a knockout mouse
embryonic stem cells homologous recombination drugs blastocysts
conditional knockout mice are important to study the fxn. of...
essential genes
T/F: cloned animals are identical to their donors in all respects
false
T/F: in gene therapy, the therapeutic gene is always a wild-type copy of a gene whose fxn. is lacking, causing a disease phenotype
false
what offered the first encouraging results with gene therapy
gene therapy to treat SCID-X1
retroviral vectors
gene therapy vectors that are partial retrovirus genomes containing a therapeutic gene; the therapeutic gene becomes stably integrated into the genomes of somatic cells.
what is introduced into animals or plants to create a GM organism?
genetic material
although some countries restrict or bar their importation, ________ _______ organisms are generally though to be safe for consumption
genetically modified
GM (genetically modified)
genetically modified crops; agricultural plants that contain transgenes.
the Monsanto Compant has produced transgenic plant strains resistant to _________, which is a herbicide. the herbicide then kills ______ but not the crop of interest.
glyphosate;weeds
a common feature of gene editing technologies is the use of a _______, either a protein or an RNA molecule to bring a DNA nuclease to a specific genomic location
guide
gene knockout allows researches to study...
how the loss of normal gene fxn. affects an organism
a primate model for what human disease carried a transgene containing an expanded CAG repeat region?
huntington disease
transgenic organism
individuals carrying a transgene.
the targeting construct for making conditional knockout mice contains two __________ (one containing a loxP and other containing a drug ressitance gene flanked by two loxP sites) and one ______ of the gene to be conditionally knocked out
introns; exon
_______ mice are especially useful for the study of diseases caused by mutations in the codons for specific amino acids
knockin
mice containing a chromosome with an amorhic allele of a targeted gene are called a ________ mice
knockout
the bacterium Bacillus thuringiensis produces toxins (BT protein) that are _______ to many caterpillars that feed on food crops, but that are generally harmless to plants and animals
lethal
several patients receiving gene therapy for SCID-X1 developed _______, which was caused by insertion of the ______ vector next to a particular gene in the patients' genomes
leukemia; retroviral
to create transgenic plants using a T-DNA plasmid vector, first A. tumefaciens are transformed with two plasmid- a T-DNA vector containing the gene to be transferred and a ______ gene, and a helper plasmid that contains ____ genes but not border sequences
marker; vir
gene targeting
method for inserting DNA into a genome that relies on homologous recombination; the DNA is targeted for insertion into a specific place in the genome by sequence similarity.
reproductive cloning
methods that produce two or more genetically identical individuals
knockout mice
mice homozygous for an induced mutation in a targeted gene; the mutation destroys (knocks out) the function of the gene.
knockin mice
mice in which a gene has been altered by targeted mutagenesis. The alteration can be a point mutation or a large insertion of DNA.
a strain of mice engineered to carry a mutation that is analogous to a disease-causing mutation in a human gene is called a mouse _____
model
changing specific genes in nearly any way desired is called targeted __________
mutagenesis
the production of human protein drugs in transgenic animals and plants is called....
pharming
processes that are caried out on human proteins that cannot always be recreated if the proteins are expressed in bacteria
phosphorylation, glucosylation, cleavage of polypeps. and proper folding
valid objections to use of GM plants in agriculture
placing considerable power in a small number of transnational agribusinesses disruption of the lives of farmers and farm communities transmission of traits from GM organisms to species in the wild
to use the CRISPR/Cas9 tech. on cells grown in culture, scientists usually transform the cells with ______ containing genes to make ______ and Cas9
plasmids;sgRNA
to produce a transgenic mouse by _______ injection, the desired DNA is injected into a ________ egg
pronuclear; fertilized
molecules used as guides for gene editing to bring a DNA nuclease to a specific genomic location
protein and RNA
what viral vector's integration can result in genome mutation and sometimes cancer in patients
retroviral vectors
adeno-associated viral vectors are often used for gene therapy because, unlike _______ vectors, their DNA constructs usually do not _________ into host cell chromosomes, and therefore do not cause genome mutation
retroviral; integrate
components of T-DNA
right border (RB), left border (LB) and gene that causes cell overgrowth
two main components of an engineered CRIPSR/Cas9 sys
sgRNA and a Cas9 polypeptide that has been altered to contain a nuclear localization signal
targeted mutagenesis
technology that enables scientists to alter any particular base pairs in the genome; includes gene targeting and CRISPR/Cas9.
in gene therapy, in order to treat a disease,...
the DNA of a gene can be altered DNA can be added to the genome
embryonic stem cells are found in
the blastocyst
gene therapy to treat Leber congenital amaurosis delivers the RPE65 gene of cells of...
the eye
pharming can refer to...
the manufacture of medical products in agricultural plants or... the production of medically important proteins in the mammary glands of livestock
somatic cell nuclear transfer
the method for reproductive cloning; the nucleus of a somatic cell replaces the nucleus of an oocyte, which is subsequently fertilized in vitro, and the zygote is introduced into the womb of a surrogate mother.
pronuclei (pronucleus)
the sperm and egg nuclei present in the same cytoplasm after fertilization in mammals.
to make transgenic eukaryotes what conditions must be met?
the transgene must be replicated and maintained as the cell divides the transgenic DNA must be introduced to one or more cells cells containing the transgene must have the ability to develop eventually into gametes
pharming
the use of transgenic animals and plants to produce protein drugs.
cloned animals and their donor are not perfectly identical in all respects because...
their mitochondrial genomes are dofferent female clones will have different patterns of X inactivation than their donors the uterine environment for each animal during development will be different
gene __________ is the manipulation of genes to treat disease
therapy
how can a transgene be introduced into cells
through a treatment that disrupts the cell wall injection of DNA into the cell via viral particles
stem cells that can give rise to any cell type are said to be
totipotent
a cloned gene from one species that is introduced into another species or another individual of the same species is called a(n)
transgene
in additional to a transgene, p element vectors contain the P element ends but not the _________ gene. they also contain a _______ gene, an example of which is the wild-type white gene
transposase; marker
T/F: pharmaceutical companies use bacteria to produce medically important proteins like insulin
true
a major difficulty in producing ________ in plants is controlling the dose of the antigen
vaccines
edible ______ produced in transgenic plants could be especially advantageous for less-developed countries, since they would need no refrigeration, needles or medical professional
vaccines
chimeric mice are mated with what type of mouse to generate a nonmosaic heterozygous progeny
wild-type
