Chapter 10 Genetic Engineering Flashcards
Genetic engineering
refers to manipulation of genetic material, whether in a test tube (in vitro) or in a living organism (in vivo) using chemical or biological means.
T or F: about 40% of the human and mouse genomes can be directly aligned with each other
True
What % of human genes have a corresponding gene in the mouse genome?
80%
two general categories of mice that are generated using genetic engineering techniques
transgenic and gene-targeted
Transgenic mice
are produced by adding a gene from one animal into another
Gene-targeted mice
are produced by altering a gene that is already in the animal.
three primary ways in which genetic engineering techniques are used to generate transgenic mice
- microinjection of the transgenic construct into a fertilized egg (which is called an oocyte or zygote)
- to introduce a transgene by use of a retroviral vector into the egg
- introduced into embryonic stem cells and then the embryonic stem cells are injected into mouse blastocysts
4 groups needed to produce transgenic animal
egg donor females,
embryo recipient females,
stud males, and
vasectomized (sterile) males.
used to superovulate a female mouse
receives two intraperitoneal injections of exogenous hormones:
1. pregnant mare serum gonadotropin (PMSG) and 2. human chorionic gonadotropin (hCG)
factors that may affect the efficiency of superovulation
the strain of mouse, the female’s age, her weight, the timing of the hormone injections, and the light-dark cycle.
Pronuclear injection
introduces the genetic components directly into an embryo
pronuclei
they are precursors of the zygote’s nucleus. The two pronuclei fuse, forming the nucleus.
transposon-mediated transgenesis
a bacterial system that can introduce the transgene in a single copy.
Embryonic stem (ES) cells
are undifferentiated cells that have the ability to develop into any kind of cell in the body
electroporation
In electroporation, a solution of the DNA is mixed with the mouse ES cells and then an electrical shock is applied. This opens pores in the cellular and nuclear membranes, so the DNA can flow into the nucleus. There, the DNA can insert itself randomly into the genome.
why inbred strains are most commonly chosen for genetic engineering
Because inbred strains are very uniform in their phenotypes, it is easier to see phenotypic changes in a transgenic or gene-targeted mouse from an inbred strain
Why each mouse determined to contain the transgene should be treated as the founder of a separate line of mice
each should have the insertion at a different site in the genome
when two hemizygous animals are mated
- 25% of the offspring will have two copies of the transgene, which makes them homozygous for the transgene
- 50% will be hemizygous for the transgene
- 25% will have no copy of the transgene
chimera
animal is a mix of cells from different animals
they will have tissues, including skin, that are derived from both the injected ES cells and the donor cells from the blastocyst
XY
male
XX
female
CRISPR/Cas9 system
(clustered regularly-interspaced short palindromic repeats/ CRISPR associated protein 9).
CRISPR is a DNA identification mechanism that is naturally occurring —it is the antiviral immune system for bacteria.
These systems rely on enzymes that can be directed to a specific target in the genome.
mosaic
If there is a variety of mutations, in both number and location, within an animal’s cells
backcrossing
To change the strain background, each generation of genetically modified mice is bred to wild-type mice from the desired strain. This allows the researcher to gradually dilute out the portions of the genome that carried over from the strain from which the ES cells were produced.
congenic strain
Once a mixed strain has been backcrossed for 10 generations, it is considered essentially genetically identical to the inbred strain, except for the desired genetic modification
mating a wild type female with hemizygous or heterozygous males
only half of the embryos on average will have the desired genotype
mating a wild type female with homozygous males
all the embryos will have one copy of the modified gene
biggest drawback with sperm cryopreservation
only half the germplasm is banked and upon recovery all the animals will be heterozygous
Cryopreservation is used to maintain the strains while:
minimizing genetic drift and contamination,
guarding against disasters, and
fulfilling the principles of the 3 Rs (replace, refine, and reduce).
pronuclei
the nuclei of the eggs shortly after fertilization
the introduction of an antibiotic resistance gene into a DNA fragment is used for what purpose?
it helps identify the cells that have integrated the transgene
hemizygous
if a transgene is integrated in one of the chromosomes of a pair
T or F chimeras that received the ES cell chromosomes have a copy of the mutated gene in every cell?
True
T or F like sperm, embryonic stem cells are haploid and always transmit the mutation
False
What are genes
Genes are segments along the strands of DNA that have specific sequences of these nucleotides which code for specific proteins.
chromosomes
long strands of DNA coiled to form individual structures
alleles
different versions of the same genes
homozygote (having homozygous alleles for that gene)
When both genes of a pair (one from each of the paired chromosomes) are the same
heterozygote (having heterozygous alleles for that gene)
If the genes at the same locus on a chromosome pair are different
wild type
If an animal does not contain the allele that the investigator is interested in
Homozygous null
means that both copies of the gene are absent or turned off. In this situation, the gene produces no product and the animal is called a knockout.
Homologous Recombination
embryonic stem cells that line the inside of a developing blastocyst (a preimplantation stage of early embryos) are collected and used to produce embryonic stem (ES) cell lines.
The DNA containing the mutated gene is inserted into these cells by a process called electroporation.
The cell integrates the mutated gene at the specific location corresponding to its own copy of that gene.
The cells containing the mutated gene are then injected into another developing blastocyst.
germline transmission
The most desirable chimera is one in which the new gene has been incorporated in the germ cells (egg and sperm)
Inbred strain nomenclature
strain name / name of person or laboratory
Outbred stain nomenclature
abbreviation of the holder : stock name
establishing inbred colonies
Foundation colony
Expansion colony
Production colony
coisogenic
Occasionally a spontaneous mutation occurs in an inbred strain. The animal with the mutation differs from other animals of that strain, usually by a single mutant gene
What percentage of pups will contain the transgene?
20%
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what is the term for an animal with a fragment of DNA that has been introduced into its genome?
transgenic
what technique can be used to maintain a specific strain of mice while minimizing genetic drift?
cryopreservation
t or f: the gene-targeted mutation is present in all cells of a chimera.
false. chimeras have a mixture of cells; some cells do not have the mutation.
what four types of mice are needed for producing transgenic animals?
egg donor females, embryo recipient females, stud males, vasectomized (sterile/dud) males
t or f: female mice become pseudopregnant when bred with a vasectomized male.
true
which generation of transgenic micee is designated as F1?
offspring of a founder mouse
a piece of DNA that will be inserted into the nucleus of a cell is called a ….
transgene
what is the term for placing pups with a mother other than the one that gave birth to them?
cross-fostering
when a mixed strain has been backcrossed for 10 generations, it is called a …. strain
congenic
when the transgene is incorporated into only one of the chromosomes on a pair, is the animal homozygous, heerozygous, or hemizygous?
hemizygous
Which of the following terms represents the hereditary information that is present in an animal?
a. Phenome
b. Proteome
c. Genome
d. Speciation
c. Genome
What best describes a transgenic animal?
a. Animal with genes from an external source
b. Animal which has an abnormal number of chromosomes
c. Animal which has been transferred as an embryo to a foster mother
d. Animal in which the cells of the gonads have undergone a mutation
a. Animal with genes from an external source
What percentage of the genes in the genome of a mouse have a corresponding gene in the genome of humans? a. 20% b. 40% c. 80% d. 100%
c. 80%
Which of the following definitions best describes a transgene?
a. The section of DNA that will be deleted in a cell
b. A gene found in the genome of a large number of species
c. The section of a DNA molecule coding for a specific protein
d. The fragment of DNA introduced into the nucleus of a cell
d. The fragment of DNA introduced into the nucleus of a cell
What is the purpose of adding an antibiotic resistance gene into a DNA fragment before inserting it in a
cell?
a. It protects the transgene from destruction by bacteria.
b. It helps identify the cells which have integrated the transgene.
c. It protects the animal receiving the transgene from bacterial infection.
d. It promotes cell division.
b. It helps identify the cells which have integrated the transgene.
What are pronuclei?
a. Nucleus of the sperm or egg cell during the process of fertilization before fusion
b. Nucleus of the sperm or egg before injection of a transgene
c. Nucleus of the sperm or egg in which DNA has been removed
d. Nucleus of the sperm or egg cell after fertilization and fusion
a. Nucleus of the sperm or egg cell during the process of fertilization before fusion
What is the method of genetic engineering most specific for introducing a transgene into a specific location on a chromosome? a. Transgene transfer b. Phenotype copy c. Pronuclear injection d. Gene-targeted mutation
d. Gene-targeted mutation
Using the pronuclear injection technique, what is the average percentage of offspring carrying the transgene? a. 5% b. 20% c. 50% d. 80%
b. 20%
Which of the following best describes the term allele?
a. The gene produced by insertion of a DNA fragment from another species
b. The same gene found on all chromosomes of a species
c. One of a pair of genes that occupies a specific site on a given chromosome
d. Different DNA fragments found on the same chromosome
c. One of a pair of genes that occupies a specific site on a given chromosome
If a transgene is integrated in one of the chromosomes of a pair, the animal is said to be:
a. heterozygous.
b. hemizygous.
c. homozygous.
d. haptozygous.
. b. hemizygous.
What does the generation called F2 represent among transgenic mice?
a. The founder of a line
b. The first generation offspring of the founder of a line
c. The second generation offspring of the founder of a line
d. The brother-sister mating of founders of a line
c. The second generation offspring of the founder of a line
What proportion of the offspring could you theoretically expect to be homozygous for the transgene if you mate two hemizygous parents? a. 0% b. 25% c. 50% d. 75%
. b. 25%
What percentage of the offspring of two homozygous parents will inherit the transgene?
a. 25%
b. 50%
c. 75%
d. 100%
. d. 100%
Which of the following best explains the fact that in gene-targeted mutation, the DNA fragment is
inserted at a specific locus on the gene?
a. The DNA fragment is inserted through an electric current that allows it to be transported easily to
the correct location.
b. The DNA fragment is sized to replace the whole gene.
c. The DNA fragment is flanked by DNA sequences that are identical to those found in the wild
type.
d. None of the above
c. The DNA fragment is flanked by DNA sequences that are identical to those found in the wild type.
Which cells are used to produce transgenic mice by gene-targeted mutations?
a. Embryonic somatic cells
b. Embryonic parent cells
c. Embryonic differentiated cells
d. Embryonic stem cells
d. Embryonic stem cells
Knockout animals are useful to determine what kind of information?
a. The replication of chromosomes containing the affected gene
b. The normal function of the affected gene
c. The location of the protein that regulates the affected gene
d. All of the above
b. The normal function of the affected gene
What is the best method of stopping the function of a specific gene?
a. Knockout technology
b. Knockin technology
c. Transgenic technology
d. Backcrossing
. a. Knockout technology
Which of the following are NOT used in the production of chimeras?
a. Blastocysts
b. Pseudopregnant females
c. Superovulated females
d. Unfertilized eggs
d. Unfertilized eggs
Which scenario applies to the production of chimeras?
a. Ova are harvested shortly after fertilization.
b. Oocytes are harvested in the 1-cell stage.
c. A DNA fragment is inserted in the pronuclei.
d. Embryos are harvested at the blastocyst stage.
d. Embryos are harvested at the blastocyst stage.
Mating a wild type mouse with a chimera having a transgene in its germ cells results in mice that will be \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ for the transgene. a. null b. homozygous c. heterozygous d. dominant
c. hemizygous
In which of the following scenarios could you be sure to obtain a mouse that is homozygous for the
transgene?
a. Mate a chimera with a wild-type mouse.
b. Mate two hemizygous parents.
c. Mate two homozygous parents.
d. It is not possible to know in advance if the offspring will be homozygous.
c. Mate two homozygous parents.
Which of the following order of steps should be followed to produce gene-targeted animals?
a. Inject female with PMSG; inject female with hCG; mate female with stud male; harvest embryo.
b. Inject stud male with PMSG; inject female with hCG; mate female with dud male; harvest
embryo.
c. Inject stud male with PMSG; mate female with stud male; inject female with hCG; harvest
embryo.
d. Mate female with dud male; inject female with PMSG; inject female with hCG; harvest the
embryo.
a. Inject female with PMSG; inject female with hCG; mate female with stud male; harvest embryo.
For producing gene-targeted animals, the dosage and timing of the hormone injections to produce
superovulation must take into consideration which of the following factors?
a. Staff schedule
b. Light:dark cycle in use at the facility
c. Location of the cage in the rack
d. Strain of the stud
b. Light:dark cycle in use at the facility
What is cross-fostering?
a. Mating animals of different strains
b. Placing pups with a female that did not give birth to them
c. Having more than one female in the cage with the pups
d. Having more than one male in the cage with the pups
b. Placing pups with a female that did not give birth to them
What causes female mice to become pseudopregnant?
a. Injection of PMSG
b. Change in the light/dark cycle
c. The presence of high levels of pheromones
d. Copulation with dud males
d. Copulation with dud males
What best describes a congenic strain?
a. The result of backcrossing for 10 generations
b. The result of consecutive matings of two hemizygous transgenic parents
c. The result of brother-sister mating of transgenic animals
d. The result of consecutive matings of two transgenic homozygous parents
. a. The result of backcrossing for 10 generations
What is the best age to genotype mice in order not to disturb the litter?
a. Right after birth
b. In the first two weeks of life
c. Around weaning time
d. When the pup reaches sexual maturity
c. Around weaning time
What technique allows a mouse strain to be taken “off the shelf” but still be available for use when needed? a. Southern blot b. Knock-in technology c. Artificial insemination d. Cryopreservation
d. Cryopreservation
When wild type females are bred to hemizygous males, what percentage of the offspring can be expected to have the transgene? a. 10% b. 25% c. 50% d. 100%
c. 50%
Which of the following processes describes recovering a mouse line?
a. The chimeras are genotyped and the heterozygous ones are mated together.
b. The founders resulting from pronuclear injection are mated to wild type to produce offspring that
have the transgene.
c. The frozen embryos are thawed and implanted in a pseudopregant female.
d. The frozen embryos are thawed and grown in vitro.
. c. The frozen embryos are thawed and implanted in a pseudopregant female.
If a mouse no longer looks pregnant and no pups are visible, the technologist can examine the mouse’s \_\_\_\_\_\_ for evidence of birthing. a. vulva b. copulatory plug c. feces d. placenta
a. vulva
Which of the following is a sign that indicates good maternal care and good pup health?
a. There is a visible milk spot in the pups.
b. Pups are scattered about the cage.
c. The pups spend a lot of time apart from the dam.
d. A couple of the pups are larger than the others.
a. There is a visible milk spot in the pups.
Which of the following can help the technologist observe the pups without disturbing the mother?
a. Use UV light instead of regular lighting.
b. Use clear nesting boxes.
c. Remove all bedding.
d. Remove the mother from the cages while observing the pups.
b. Use clear nesting boxes.
Which type of study has been made possible by advances in the field of genetic engineering?
a. Elucidating the function of specific genes
b. Developing animal models for human genetic diseases
c. Tailoring drugs to a particular patient
d. All of the above
. d. All of the above
An experiment performed in vitro is one done:
a. in living organisms.
b. using a questionnaire to survey opinion.
c. using glass syringes to collect blood.
d. in a test tube.
d. In a test tube
Why is using inbred strains preferred over outbred strains to create transgenic mice?
a. Inbred strains are easier to maintain in colonies.
b. Inbred strains have uniform phenotypes so differences are easier to see.
c. Inbred strains have greater reproductive efficiency.
d. Inbred strains have more chromosomes so it’s easier to integrate an extra transgene.
b. Inbred strains have uniform phenotypes so differences are easier to see.
In the pronuclear injection technique, some females are mated to vasectomized males to:
a. produce more eggs.
b. induce pseudopregnancy.
c. increase the probability that the transgene will be incorporated in the cells.
d. provide sperm for fertilizing the ova.
b. induce pseudopregnancy.
In performing genetic analysis, which of the following is obtained from the animal and analyzed to
determine if the transgene is present in the animal’s cells?
a. RNA
b. DNA
c. Protein
d. Mitochondria
b. DNA
What is the correct genetic designation for the initial founder of a line of transgenic mice?
a. F0
b. F1
c. N0
d. N1
. a. F0
What is the term for an animal with a copy of the transgene on one chromosome but not on the other chromosome of the pair? a. Homozygous b. Heterozygous c. Hemizygous d. Haptozygous
c. Hemizygous
What term best describes embryonic stem cells?
a. Multireactive
b. Undifferentiated
c. Undivided
d. Multinuclear
Undifferentiated
