FAQ

By fusing the ligand binding region (LBD) of the human estrogen receptor (ER) with Cre recombinase, a fusion protein (Cre-ER) localized in the cytoplasm can be generated. Only upon estrogen induction, the fused Cre protein can dissociate from the anchor protein HSP90 via conformational changes and enter the nucleus, where the Cre protein recognizes the loxP site and undergoes recombination. In this way, by controlling the injection time of estrogen, the time-specific regulation of gene recombination can be achieved.

In order to avoid the interference by endogenous estrogen, a point mutation (G521R) can be generated in the ligand binding region, thus allowing Cre-ER to only respond to the induction by exogenous synthetic estrogens (e.g., Tamoxifen, 4-OHT). Such fusion protein is named as Cre-ERT. Another fusion protein carrying LBD mutation, the well-known Cre-ERT2, was shown to be much more sensitive to 4-OHT than Cre-ERT. It carries three point mutations in the human ER LBD: C400V/M453A/L544A.

Figure 6. Schematic diagram of Tamoxifen-induced activation of Cre. (The picture is from Inducible CreMice. Methods in Molecular Biology 530:343-63 February 2009).

Conditional knockout mice (also known as Flox mice) are mice whose target genes contain paired loxp sites. After mating them with Cre mice, target genes can be knocked out in specific tissues or cells.

Option One

If Flox mice are mated with Cre mice widely expressing Cre or expressing Cre only in germ cells, systemic KO mice can be obtained. The breeding procedure is shown in the figure. In the ES targeting vector, the screening gene neo is located between two loxp sites and it is not necessary to remove neo separately. The structure is 5' arm-loxp-flox-frt-neo-frt-loxp-3' arm and the neo gene can be simultaneously removed by directly mating with mice expressing Cre in the gonad.

Figure 3. Breeding procedure to obtain systemic and homozygous KO mice from conditional knockout mice.

Option Two

The procedure shown in Figure 4 can be adopted if breeding is carried out by mating with mice showing tissue-specific Cre expression. In general, heterozygous flox mice transfected with Cre recombinase are mated with heterozygous flox mice to obtain homozygous conditional knockout mice carrying integrated Cre enzyme. At this time, homozygous flox mice in the same litter that do not carry integrated Cre enzyme are generally selected as the control group for the experiment.

Figure 4. Breeding procedure 1 to obtain tissue-specific knockout mice.

Option Three

The protocol in Figure 5 can also be used to increase the proportion of mice in the experimental group.

Figure 5. Breeding procedure 2 to obtain tissue-specific knockout mice.

For knockout or genetically mutated mice with an inbred background, mating between heterozygous knockout mice is generally carried out to obtain homozygous knockout or genetically mutated mice. At this time, wild-type or heterozygous mice born in the same litter are generally selected as the control group for the experiment.

Figure 2. Breeding procedure for systemic knockout mice.

Removal of neo from knockout mice

If the removal of neo may cause a complete loss of gene functions and if there are no other genes in the vicinity of the genome, the neo gene does not have to be removed. On the other hand, if only a certain functional domain is knocked out, some proteins with partial functions may appear. Alternatively, there may be other genes located around the knockout gene. In these two cases, it is suggested to remove the neo gene to avoid the occurrence of a phenotype not consistent with the gene knockout. If there is sufficient time, it is suggested to remove the neo gene to avoid the occurrence of other phenotypes caused by neo.

After the Founder mice are obtained, a transgenic strain can be established according to the following procedure.

Figure 1. Procedure to establish a mouse strain with a randomly inserted transgene.

Notes about transgenes mediated by the Piggybac transposase system: 

The Piggybac transposase tends to insert the target fragment into a transcriptionally active region, thus greatly increasing the probability of obtaining Founder mice with positive expression of the target gene. However, the insertion of transgenes mediated by the Piggybac transposase system may cause gene integration at multiple sites.Due to the existence of integration at multiple sites, the following phenomena will occur during the subsequent breeding of Founder mice along with the separation of multiple integration sites: 1. The proportion of mice with positive results of genotype identification is higher than that of single site insertion; 2. In progeny mice with positive expression of the target gene, the level of expression is lower than that in the Founder mice (the amount of reduction is dependent on the number and location of integration sites in Founder mice) and is inconsistent among different progeny mice.Therefore, it is recommended that each Founder mouse be backcrossed with wild-type mice for at least 2–3 generations to obtain transgenic mice with single site integration and stable expression of the exogenous gene.

Sentinel mice mainly refer to a type of mice with a healthy background that are introduced into important breeding groups. Through direct contact with the target mice or indirectly contact with dirty litter, the inspection of pathogenic microorganisms in the breeding area can be tested using sentinel mice. In addition, sentinel mice can be used to substitute important breeding groups to undergo medical examinations. 

In general, healthy mice (excluding immunodeficient mice) that are older than 4 weeks and not contaminated by specific pathogens can be selected as sentinel mice. The strains of sentinel mice, such as ICR (CD1), BALB/c, FVB, and C57BL/6, can be selected as needed. Sentinel mice are usually housed at a density of 2–3 mice per cage. In addition, they are placed in the lowest position on the cage shelf. Typically, a cage of sentinel animals is used to monitor case shelves housing 60–100 cages. The litter used for the sentinel mice is the dirty litter that has been used by other mice. About 30–50 g litter is placed in each cage and the sentinel mice must live on the dirty litter for at least 4 weeks. 

In general, the national standard stipulates that three months is counted as a testing cycle. Through the examination of sentinel mice, the condition of pathogens in the environment can be monitored in real time without affecting the experimental animals, thus ensuring standardized health and quality of experimental animals as well as repeatable experimental results. The examination of sentinel animals also has its limitations. For example, airborne pathogenic microorganisms not transmitted by contact may be overlooked, such as Sendai virus and Pasteurella pneumophila. Therefore, it is recommended to also examine random animals.

Check for the following problems when the number of offspring is decreased in the breeding process or when no offspring is born:

The breeding environment is too noisy: during the breeding and feeding of mice, noise should be minimized to ensure that the mice live in a quiet environment.

Pregnant mice are stimulated by external stress: The operation and environmental changes incurred to pregnant mice should be minimized. When pregnant mice and lactating mice are stimulated by external stress, they may eat or abandon their cubs.

The mating time between male and female mice is too short: Ensure an appropriate length of mating time between male and female mice to increase the chance of mating. In addition, do not remove mating male mice from the cage when pregnant mice are in labor, and do not put the male mice back into the breeding cage before the offspring mice are weaned.

Increase the darkness of the environment: avoid unnecessary light in the night rhythm because it can affect the circadian rhythm of the mice.

The mice are unable to build nests: Provide soft fiber materials, such as cotton wool, paper towels or cork flocculus, in cages to facilitate the establishment of a safe and comfortable environment for labor, thus increasing the number of litters.

Nutritional deficiency: If the lipid component is too high or too low in mouse diet, or if the protein intake is insufficient or excessive, the reproduction of mice will be affected.In addition, the source of tocopherol, such as seeds and nuts, can be provided as appropriate.

In general, at least 2–4 pairs of mice at the breeding age are required for strain breeding. If the mice of a special strain have birth defects or require accelerated breeding, consider increasing the number of mating pairs. Alternatively, an IVF rapid breeding method can be used. In this case, only 2–3 fertile male mice are needed.

An animal strain established by 20 (or more) consecutive generations of mating among siblings (or via mating between a parental generation and a progeny generation). The inbreeding coefficient of the stain should be greater than 99%, and all individuals in the strain should be traced back to a common pair of ancestors.

Characteristics: The genetic background is uniform and the experimental results are consistent. However, due to inbreeding depression and low viability, an inbred strain has a low number of litters and a more stringent demand for the breeding environment and nutritions.

The commonly used C57BL/6, Balb/c, and FVB all belong to inbred strains.

SPF is the abbreviation of "Specific-pathogen-free" and represents animals which do not carry specific pathogens and parasites that may interfere with the experiment. SPF animals should be housed in a barrier system, and are subject to strict microbial and parasite control. As internationally recognized and standard laboratory animals, SPF grade animals can be widely used in life science and medical research experiments.

The full name of AAALAC is the Association for Assessment and Accreditation of Laboratory Animal Care. AAALAC International certification is a symbol for the quality and biosafety standard of experimental animals. As a sign of quality suitable for internationally leading medical research, AAALAC International certification represents the true commitment to humane care of animals and is a prerequisite for organizations involved in the application and production of experimental animals to participate in international exchanges and competition.