Transgenic animals: production, maintenance and applications MCQs With Answer

Introduction: Transgenic animals: production, maintenance and applications MCQs With Answer is designed for M. Pharm students preparing for advanced pharmacological and toxicological screening courses. This collection presents focused multiple-choice questions that cover core techniques—pronuclear injection, embryonic stem cell manipulation, somatic cell nuclear transfer, and modern genome editing with CRISPR/Cas9—plus colony management, genotyping, phenotyping, and ethical/regulatory aspects. Questions emphasize mechanistic understanding, experimental design, troubleshooting, and practical applications in drug discovery and toxicology. Each item targets knowledge required for creating, validating and applying transgenic models to evaluate pharmacokinetics, target engagement, toxicity, and biomarker development in preclinical research.

Q1. Which method is most commonly used for creating random integration transgenic mice by inserting DNA into fertilized zygotes?

• Embryonic stem (ES) cell homologous recombination
• Pronuclear microinjection into zygotes
• Somatic cell nuclear transfer (cloning)
• Viral transduction of adult tissues

Correct Answer: Pronuclear microinjection into zygotes

Q2. Which technique historically enabled targeted gene knockout in mice via homologous recombination?

• Pronuclear microinjection
• CRISPR/Cas9-mediated non-homologous end joining
• Embryonic stem (ES) cell-based gene targeting
• Somatic cell nuclear transfer

Correct Answer: Embryonic stem (ES) cell-based gene targeting

Q3. In the Cre-loxP system used for conditional gene manipulation, what is the role of loxP sites?

• They encode the Cre recombinase enzyme
• They are recognition sites where Cre mediates site-specific recombination
• They act as promoters to drive transgene expression
• They function as antibiotic selection markers

Correct Answer: They are recognition sites where Cre mediates site-specific recombination

Q4. Which inducible system allows reversible regulation of transgene expression in response to tetracycline or doxycycline?

• Cre-loxP inducible system
• Tet-On/Tet-Off system
• FLP-FRT recombinase system
• RNA interference (RNAi) only

Correct Answer: Tet-On/Tet-Off system

Q5. What is the primary advantage of CRISPR/Cas9 over traditional ES cell targeting?

• It always avoids off-target effects
• It requires selection markers for integration
• It enables rapid, programmable site-specific DNA cleavage guided by sgRNA
• It only creates random integrations

Correct Answer: It enables rapid, programmable site-specific DNA cleavage guided by sgRNA

Q6. Mosaicism in founder animals produced by zygote injection most commonly arises because:

• The transgene integrates into mitochondrial DNA only
• The transgene integrates after the first few embryonic cell divisions
• All cells receive identical transgene copies at fertilization
• Transgene expression is silenced epigenetically in founders

Correct Answer: The transgene integrates after the first few embryonic cell divisions

Q7. Which reporter gene is most frequently used for real-time fluorescent imaging of transgene expression in tissues?

• Beta-galactosidase (lacZ)
• Firefly luciferase
• Green fluorescent protein (GFP)
• Neomycin resistance gene (neo)

Correct Answer: Green fluorescent protein (GFP)

Q8. A typical outcome of pronuclear microinjection is:

• Insertion at a single defined genomic locus with one copy number
• High frequency of precise homologous recombination
• Random insertion often with variable copy number and concatemer formation
• Guaranteed germline transmission in all founders

Correct Answer: Random insertion often with variable copy number and concatemer formation

Q9. What does the term “knock-in” most accurately describe?

• Complete deletion of a gene from the genome
• Insertion of a specific sequence at a defined genomic locus
• Random integration of a transgene into the genome
• Temporary suppression of a gene using siRNA

Correct Answer: Insertion of a specific sequence at a defined genomic locus

Q10. How is germline transmission of a transgene usually confirmed experimentally?

• Sequencing only the founder animal’s somatic tissues
• Observing phenotype in the founder without breeding
• Breeding founders and genotyping their offspring for presence of the transgene
• Measuring transgene mRNA in cultured cells from the founder

Correct Answer: Breeding founders and genotyping their offspring for presence of the transgene

Q11. Which molecular technique is traditionally considered most reliable for determining transgene copy number and insertion pattern?

• Conventional PCR with endpoint detection
• Southern blot analysis
• Western blot for transgene protein
• In situ hybridization on tissue sections

Correct Answer: Southern blot analysis

Q12. Which strategy reduces off-target editing when using CRISPR/Cas9 in animal embryos?

• Using very long single guide RNAs (>30 nt) without validation
• Delivering high concentrations of Cas9 protein only
• Using high-fidelity Cas9 variants and carefully designed sgRNAs with off-target prediction
• Relying solely on phenotypic screening without sequencing

Correct Answer: Using high-fidelity Cas9 variants and carefully designed sgRNAs with off-target prediction

Q13. Which mouse strain’s ES cells were classically preferred for generating germline-competent chimeras?

• C57BL/6 ES cells exclusively
• 129 strain ES cells due to high germline competency
• Balb/c ES cells because they never require selection
• Outbred stocks because they increase targeting efficiency

Correct Answer: 129 strain ES cells due to high germline competency

Q14. For in vivo non-invasive bioluminescent imaging of transgenic animals, which reporter gene is optimal?

• Green fluorescent protein (GFP)
• Beta-galactosidase (lacZ)
• Firefly luciferase
• Puromycin resistance gene

Correct Answer: Firefly luciferase

Q15. Somatic cell nuclear transfer (SCNT) is primarily used to produce animals that are:

• Randomly mutagenized by chemical agents
• Genetically identical clones of the nuclear donor
• Produced with targeted CRISPR edits only in germ cells
• Created by fertilizing two genetically different gametes

Correct Answer: Genetically identical clones of the nuclear donor

Q16. Which selectable marker is commonly used to select genetically modified embryonic stem cells in culture?

• Green fluorescent protein (GFP)
• Neomycin phosphotransferase (neo) conferring G418 resistance
• Firefly luciferase
• Hygromycin B acetyltransferase always used without antibiotics

Correct Answer: Neomycin phosphotransferase (neo) conferring G418 resistance

Q17. A “floxed” allele refers to a genomic region that is:

• Deleted permanently in all tissues
• Flanked by loxP sites allowing conditional excision by Cre
• Tagged with GFP for visualization
• Inserted at a random genomic location

Correct Answer: Flanked by loxP sites allowing conditional excision by Cre

Q18. When selecting founder lines for long-term studies, which criterion is most critical?

• Presence of transgene only in somatic cells of the founder
• High mosaicism in multiple tissues of the founder
• Stable germline transmission and a well-characterized insertion site/copy number
• Any founder that displays the expected phenotype without genotyping

Correct Answer: Stable germline transmission and a well-characterized insertion site/copy number

Q19. For pharmacological and toxicological evaluation using transgenic animals, phenotyping should primarily include:

• Only behavioral tests unrelated to drug exposure
• Assessment of pharmacokinetics, tissue distribution, target engagement and toxicity endpoints
• Genotyping once and no further monitoring
• Only histology without biochemical or functional assays

Correct Answer: Assessment of pharmacokinetics, tissue distribution, target engagement and toxicity endpoints

Q20. Best practices for maintaining transgenic mouse colonies and preserving strain integrity include:

• Continuous closed breeding without any record-keeping
• Periodic backcrossing to defined background, genetic monitoring, and cryopreservation of sperm/embryos
• Allowing random outcrossing to reduce workload
• Never genotyping to avoid disturbing animals

Correct Answer: Periodic backcrossing to defined background, genetic monitoring, and cryopreservation of sperm/embryos

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