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Flashcards in LAM28_SG Deck (45)
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The mouse remains the primary choice for transgenic experimentation due to these 2 reasons:

relative ease of embryo/adult manipulation, and unparalled depth of murine genetic knowledge


DNA sequences that can be transferred into the mouse genome randomly are called:

classical transgenic


DNA sequences that can be transferred into the mouse genome in a targeted fashion are called:

knockout transgenic


When a transgene-bearing mouse expresses a functional transgenic protein, a new ___________ may be apparent.



Knockout mice must be bred to ____________ in order to fully evaluate the phenotype.

homozygosity (null genotype)


True/False: The degree of expression and/or detection of a transgene may differ significantly when placed into different inbred and non-inbred genetic milieus.



List 3 criteria that should be reviewed/considered before proceeding with a transgenic mouse program:

genetic background (is it critical to the experiment?)behavioral characteristics of the parent strains (aggression, cannibalization, mothering) reproductive characteristics of the potential parental strains (litter size, response to superovulatory stimulation, gestation time predictability)normal pathologies of the parent strains being consideredcoat color (important or useful)minimal acceptable health status (for project & w/i facility)


Name the 3 inbred strains most frequently used in transgenic research:

C57BL/6, FVB/N, and 129/SvEv


Pronulcei of zygotes of this strain are somewhat larger and more distinctive than those collected from comparable strains, making them good for microinjection.



ES cell lines derived from this strain are reputed to have a high incidence of germline transmission and are easily maintained in culture.



Name the 4 types of colonies necessary to produce transgenic mice:

embryo donor female mice (superovulated), fertile stud male mice (proven breeders), embryo transfer recipient female mice (pseudopregnant), sterile stud male mice (vasectomized)


Name 5 easily avoided potential depressors of breeding success and embryo yield

use of cold superovulatory hormone solutions, unscheduled interruptions in the light/dark phases of the light cycle in the room, use of females less than 48 h after arrival on site, cage-rack machinery vibrations or any type of repeated loud noises, excessive or rough handling, especially after mating or during the dark phase, bedding change within 24h after breeding, housing of rats or nonrodent species within the room, sacrifice of animals within the room, continued presence of multiple males within the mating cage, viral or bacterial infections (may be subclinical)


What are the two hormones used to induce superovulation and/or timed pregnancy?

pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG)


At what percentage of unusable oocytes should the environment and culture media be evaluated for potential problems?



One proven breeder stud male should be maintained for every ___ pronuclear embryos or ___ blastocysts required daily.

7, 3


What is the absolutely critical factor in achieving satisfactory results with the embryo transfer of manipulated embryos?

the skill of the animal surgeon


The tactile and olifactory stimuli of mating with sterile males will elicit pseudopregnancy in ___ to ____ % of the recipient females.



The selection of recipient females with different coat color genetics than that of the transferred embryos is suggested for what reason?

for quality control


When should the pseudopregnant female be mated in comparison to the developmental age of the transferred embryos? Why?

One day later. This asynchrony compensates for the delayed embryo development attributable to manipulation in vitro


How long should a foster cage be undisturbed or cleaned after transfer of pups?

1 week


How many vasectomized males should be available compared to the number of pseudopregnant females desired per week?



Name the four major support systems that should be established before a transgenic line is created or imported into a facility:

an established facility health program, explicit and well-documented breeding strategy standard operating procedures and guidelines, a fastidious record-keeping system for reproductive and phenotypic data, access to an internal or external embryo cryopreservation program


True/False: health risks within a transgenic facility are more challenging than those within a nontransgenic facility?



What is the purpose of backcrossing a transgenic to an inbred strain?

stabilization of the transgene on a specific genetic background


Name 3 goals of a transgenic breeding program:

stabilization of the transgene on a specific genetic background by backcrossing to an inbred strain, breeding to transgenic homozygosity, utilization of sibling crosses to stabilize genetic and phenotypic expression, expansion of the colony to provide animals for research, production of embryos for cryopreservation, surgical rederivation of the line


True/False: Phenotypic evidence is predictive of germline transmission.



True/False: Background strain can be very important to the expression of the transgene.



When a breeding unit is being set up, how long should the male be placed in the cage before the entry of the females?

24-48 hours


Why may homozygosity not always be realized in transgenic development?

potential homozygous lethality or infertility resulting from certain transgenes


What are the most reliable methods of genotypic analysis?

molecular analyses of DNA (Southern blot hybridization assay or polymerase chain reaction- PCR)


True/False: After genotypic analysis confirmation of zygosity, subsequent confirmation of zygosity by test mating is highly recommended.



What is the minimum number of offspring from each transgenic animal that should be evaluated in order to confirm homozygosity?



Give 2 examples of problems that may be associated with the integration locus and/or knockout phenotypes:

abnormal immune responses, altered life span, sex-influenced survival, reduced litter size


What two characteristics of transgenic lines may require stringent barrier procedures and specially prepared husbandry supplies?

Altered immune competence and associated environmental requirements of certain lines


It is possible for multiple copies of the transgene to integrate at more than one locus in animals created by what process?

pronuclear microinjection


Name 3 critical items for a transgenic nomenclature system:

designation of specific background strains or stocks used, the exact transgene construct, any information regarding integration or insertion, the laboratory of origin, the method of transgenesis, a complete backcrossing history, the lineage designation


Name the 4 parts of the ILAR transgenic nomenclature system:

1. Tg (i.e. transgene) plus 1-3 letters to designate the mode of DNA insertion (i.e. N, nonhomologous; R, retroviral vector; H, homologous recombination), 2. specific DNA insertion information (not to exceed 8 letters and in parentheses), 3. laboratory-assigned number (the unique number assigned by the laboratory of origin to each confirmed stable insertion with germline transmittance), 4. unique laboratory code (maintained by ILAR)


Cryopreserved embryos are protected from what three problems?

genetic mutation and drift, genetic contamination, and potential transgene rearrangement.


Why is cryopreservation of the pronuclear-stage and 1-celled embryos challenging?

because of frequent damage to critical cytoskeletal structures and the more varied timing of these embryos at collection


Why is cryopreservation of blastocysts even more difficult?

because of the dynamics of the fluid-filled blastocoele cavity


What is the major challenge to the survival of frozen embryos?

the natural formation of intracellular ice crystals


Explain how cryoprotectants avoid/minimize this problem.

Cryoprotectants permit the controlled dehydration of the embryo by directly lowering the freezing point of the cell as the extracellular fluid is cooled. As the embryonic cells lose water and shrink, the formation of intracellular ice is avoided.


Why must cryoprotectants be removed from the embryonic cells as quickly as possible before the cells become metabolically active?

cryoprotectants are toxic


What is the expected survival and recovery rate range for 8-cell-stage embryos?



What concept allows for the patenting or licensing of transgenic animals?

intellectual property rights