LAM28_SG

Question 1

The mouse remains the primary choice for transgenic experimentation due to these 2 reasons:

Answer 1

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

Question 2

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

Answer 2

classical transgenic

Question 3

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

Answer 3

knockout transgenic

Question 4

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

Answer 4

phenotype

Question 5

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

Answer 5

homozygosity (null genotype)

Question 6

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.

Answer 6

TRUE

Question 7

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

Answer 7

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)

Question 8

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

Answer 8

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

Question 9

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

Answer 9

FVB/N

Question 10

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

Answer 10

129/Sv

Question 11

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

Answer 11

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

Question 12

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

Answer 12

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)

Question 13

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

Answer 13

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

Question 14

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

Answer 14

25%

Question 15

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

Answer 15

7, 3

Question 16

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

Answer 16

the skill of the animal surgeon

Question 17

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

Answer 17

50-80

Question 18

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

Answer 18

for quality control

Question 19

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

Answer 19

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

Question 20

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

Answer 20

1 week

Question 21

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

Answer 21

half

Question 22

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

Answer 22

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

Question 23

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

Answer 23

TRUE

Question 24

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

Answer 24

stabilization of the transgene on a specific genetic background

Question 25

Name 3 goals of a transgenic breeding program:

Answer 25

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

Question 26

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

Answer 26

FALSE

Question 27

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

Answer 27

TRUE

Question 28

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

Answer 28

24-48 hours

Question 29

Why may homozygosity not always be realized in transgenic development?

Answer 29

potential homozygous lethality or infertility resulting from certain transgenes

Question 30

What are the most reliable methods of genotypic analysis?

Answer 30

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

Question 31

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

Answer 31

TRUE

Question 32

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

Answer 32

10

Question 33

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

Answer 33

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

Question 34

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

Answer 34

Altered immune competence and associated environmental requirements of certain lines

Question 35

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

Answer 35

pronuclear microinjection

Question 36

Name 3 critical items for a transgenic nomenclature system:

Answer 36

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

Question 37

Name the 4 parts of the ILAR transgenic nomenclature system:

Answer 37

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)

Question 38

Cryopreserved embryos are protected from what three problems?

Answer 38

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

Question 39

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

Answer 39

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

Question 40

Why is cryopreservation of blastocysts even more difficult?

Answer 40

because of the dynamics of the fluid-filled blastocoele cavity

Question 41

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

Answer 41

the natural formation of intracellular ice crystals

Question 42

Explain how cryoprotectants avoid/minimize this problem.

Answer 42

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.

Question 43

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

Answer 43

cryoprotectants are toxic

Question 44

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

Answer 44

60-90%

Question 45

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

Answer 45

intellectual property rights