Breeding and genotyping a mouse line

A mouse line is a group of mice that carry the same engineered change in their DNA and are bred to keep it. In a knockout line, part of a gene has been removed or replaced so that the gene no longer works. Every mouse has two copies of each gene, one from each parent, so a mouse can carry two normal copies (wild type, written +/+), one normal and one knockout copy (heterozygous, +/−) or two knockout copies (homozygous knockout, −/−). The genotype cannot be seen by looking at the mouse: two black mice in the same cage can carry different genotypes.

Each parent passes one of its two copies to each pup, chosen at random. This is Mendelian inheritance: when two heterozygous mice are crossed, on average one quarter of the pups are +/+, one half +/− and one quarter −/−. Breeders are chosen so that the litters contain the mice the study needs, ideally knockout mice and wild-type littermates that share parents, cage and age.

To find out which genotype each pup carries, a small biopsy (usually the piece of ear taken when the mouse is ear-notched for identification) is lysed to release DNA, and a short region of the gene is copied millions of times by the polymerase chain reaction, PCR, using a heat-stable DNA polymerase (Saiki et al., 1988). The primers are designed so that the normal and the knockout copy give products of different length, which are separated by size on an agarose gel. A wild-type mouse shows one band, a homozygous knockout another, and a heterozygote both.

Genotyping only works when every step is done carefully: tubes must be labelled correctly, controls must be included in every run, and the results must be written back to the right cage card. The simulation knows the true genotype of every pup, so the consequence of each error can be shown.

Learning objectives

• Explain wild-type, heterozygous and homozygous knockout genotypes and predict the offspring of a cross with a Punnett square
• Choose breeders that give knockout mice with littermate controls, and plan a breeding cage, plug check and weaning
• Identify weaned mice by sex and ear notch code and keep biopsy tubes matched to the mice
• Set up a genotyping PCR with a correct master mix and the four controls, and choose a suitable annealing temperature
• Read a genotyping gel against a ladder and controls, recognise an invalid run, and assign genotypes
• Compare genotype counts with the Mendelian expectation and choose experimental and control mice with the 3Rs in mind

Ethics and safety

Breeding and biopsy of mice are regulated procedures. They require approval by an Institutional Animal Ethics Committee; in India such work is governed by the CCSEA under the Prevention of Cruelty to Animals Act 1960, with equivalent frameworks in the European Union (Directive 2010/63/EU), the United States (Animal Welfare Act and PHS Policy) and the United Kingdom (Animals (Scientific Procedures) Act 1986). Ear notching for identification is done by trained staff following the approved protocol. Breeding programmes are planned so that no more animals are bred than the study needs (Reduction), surplus animals are recorded and used or managed as the approval allows, and experiments are reported following the ARRIVE guidelines, developed by the NC3Rs in the United Kingdom and endorsed by journals internationally (Percie du Sert et al., 2020). PCR and gel work need laboratory safety training: nucleic acid stains and UV light are hazards, so gloves and eye or face protection are worn and stains are handled as the local risk assessment requires.

Key terms

• Allele: One version of a gene. Here the wild-type allele (+) is the normal gene and the knockout allele (−) is the engineered copy that no longer works.
• Genotype: The two alleles a mouse carries: +/+ (wild type), +/− (heterozygous) or −/− (homozygous knockout).
• Knockout: A gene inactivated on purpose, here by replacing one exon with a selection cassette.
• Exon: A part of a gene that is kept in the messenger RNA and usually codes for protein; exons are separated by introns.
• Littermates: Pups born in the same litter. Littermate controls share parents, cage and age with the experimental mice.
• Vaginal plug: A firm, whitish mass of coagulated seminal fluid left in the vagina after mating; it shows that mating took place, not that the female is pregnant.
• Weaning: Separating pups from their mother, at about three weeks of age in mice (approximate and strain-dependent).
• Anogenital distance: The distance between the anus and the genital papilla, longer in males; it is used to sex young mice.
• Ear notch code: A pattern of small notches in the ears that gives each mouse a number; the removed tissue can be used for genotyping.
• PCR: The polymerase chain reaction: repeated cycles of heating and cooling in which a heat-stable polymerase copies the DNA between two primers.
• Primer: A short single-stranded DNA molecule that binds next to the region to be copied and gives the polymerase a starting point.
• Master mix: One tube containing everything common to all reactions (buffer, magnesium, nucleotides, primers, polymerase and water), shared out before each sample is added.
• No-template control: A reaction with water instead of DNA. Any band in it means contamination, and the whole run is rejected.
• DNA ladder: A mixture of DNA pieces of known size run beside the samples so that band sizes can be read off the gel.