생쥐 지노타이핑
View the full transcript and gain access to JoVE Science Education videos
Q1: What is the difference between homozygous wildtype, heterozygous, and homozygous knockout mice?
Homozygous wildtype mice have two functional gene copies and normal phenotype. Heterozygous mice carry one functional and one disrupted copy. Homozygous knockout mice have both gene copies disrupted, allowing researchers to study the gene's full functional role. These genotypes are critical for understanding how specific genetic changes affect mouse biology and development and reproduction of the laboratory mouse.
Q2: How do you extract DNA from mouse tissue for genotyping?
Collect a small tissue sample (2-5 mm of tail) and digest it overnight in lysis buffer containing proteinase K enzyme. Centrifuge to pellet hair and debris, then add alcohol to precipitate nucleic acids. Centrifuge again to collect the DNA pellet, wash with 70% ethanol to remove salts, and resuspend in water or buffer for genotyping analysis.
Q3: What are transgenic mice and why are they used in research?
Transgenic mice carry inserted DNA fragments called transgenes introduced into their genome. A common transgene drives expression of green fluorescent protein (GFP) from jellyfish using tissue-specific promoters, allowing researchers to easily identify cells from specific tissues by green fluorescence. This enables tracking of gene expression patterns and cellular behavior in living organisms.
Q4: How do you interpret PCR-based genotyping results on an agarose gel?
Compare band patterns to control samples. Wildtype controls show only the wildtype band size; transgenic controls show only the transgene band size; heterozygous controls display both bands. Unknown mice with one wildtype band are homozygous wildtype, one transgene band indicates homozygous transgenic, and two bands indicate heterozygous genotype.
Q5: Why is it important to label mice before genotyping?
Labeling mice with permanent identifiers like ear notches ensures researchers can track individual animals after genotyping and match them to their genetic results. This allows scientists to identify and select mice with desired genotypes for experiments and maintain accurate records across multiple generations of breeding.
Q6: What are some practical applications of mouse genotyping in research?
Genotyping identifies offspring carrying multiple transgenes needed for complex genetic models, such as cancer studies requiring both an oncogene and Cre recombinase. It also enables retrospective analysis when direct embryo sampling is impossible, such as measuring embryonic heart rate while preserving fetal development by collecting tail biopsies after ultrasound measurements.
Q7: What precautions should you take when collecting tissue samples from multiple mice?
Mark used segments of the blade or scissors to prevent cross-contamination between samples. Using the same part of the cutting tool on multiple animals can transfer DNA from one mouse to another, compromising genotyping accuracy and leading to false or ambiguous results in your analysis.