Mouse Breeding


We hope that this information will be useful to investigators who are unfamiliar with mouse breeding or who are breeding transgenic or gene targeted mice for the first time. These suggestions are based on our experience. They are open to modification and should not be construed as a comprehensive set of rules.

Mouse Breeding Recommendations

1. Keep accurate breeding records. Make a pedigree for each transgenic founder or embryonic stem cell-mouse chimera.

2. Mate mice when they are sexually mature (6 to 8 weeks old). We recommend that transgenic founders or chimeras be mated to C57BL/6 mice. After 6 generations of mating to C57BL/6, more than 99% of the genetic background will be C57BL/6. By analyzing gene expression on a C57BL/6 background, any influence of the genetic background on gene expression will be controlled for by comparison to normal C57BL/6 mice. Alternatively, chimeras can be mated with 129/Sv+Tyr-c+p mice, which have the same genotype as the embryonic stem cells. This will produce mice with the targeted gene mutation on the 129/Sv+Tyr-c+p background for comparison with the mutation on the C57BL/6 background.

3. Expect litters within a month of mating since female mice go into estrus every 3 or 4 days and the gestation time of mice is 19-21 days. If no litters are produced after one month you should replace the mice that you are mating with your founder. It is possible that the transgenic founder may be infertile due to consequences of transgene expression or unknown reasons. It is possible that phenotypically male chimeras may be infertile because they are the result of colonization of a female embryo by male embryonic stem cells.

4. Reasons to use C57BL/6 mice.

a) C57BL/6 is a standard inbred strain, commonly used in transgenic breeding
b) mate 6 – 8 week-old mice for best reproductive performance
i. replace males when they are 1 year old
ii. replace females after 6 litters or when they are 6 months old
c) mate a founder male with 2 females to get 2 litters in close succession
d) mate a founder female with 1 male
e) mice usually mate again on the day the female gives birth, resulting in a second litter 3 weeks
after the first.
f) to rapidly produce animals, rotate 2 females through a male’s cage every 1- 2 weeks
g) house pregnant females 1 or 2 per cage to prevent crowded cages

5. Common problems and solutions:

a) female may not care for first litter
add proven breeder female to cage as helper and try again
b) female doesn’t care for any litter
most often seen with 129 females kept on bedding which precludes the construction
elaborate “subterranean” nests
129 mating cages should include nesting material at all times
b) male may cannibalize litter
remove male from mating cage before female gives birth
c) fighting mice
i. separate the fighting mice, house them 1 per cage if necessary
ii. females do not typically fight
iii. males may fight in the following circumstances:
male is placed in a cage containing other male(s)
male is separated at weaning and then reunited with male littermates
male is weaned into a cage that contains males from another litter
males are aggressive and may begin to fight for no apparent reason
adult male attacks immature female when female is placed in male’s cage

6. Schedule for ear tagging, tail biopsies, weaning, and mating:

a) record births on the cage card and the pedigree
b) ear tag the pups when they are two weeks old
c) obtain tail biopsies as you apply ear tags
d) isolate tail DNA and determine genotypes before pups are 21 days old
e) record genotypes in the pedigree
f) wean pups when they are 21 days old
i. remove the pups from their mothers
ii. discard un-needed non-transgenic pups
iii. house males and females separately
f) when mice are 6 weeks old they may be mated (see 1. above)

ES Cell-Mouse Chimera Breeding

Based on our experience, we propose the following guidelines for chimera breeding. If you have 10 or so chimeras to take through breeding then these guidelines apply. If you only have one or two animals then breed them indefinitely until you can produce additional animals from independent ES cell clones. You may be lucky get germline transmission at low efficiency (defined as 1 agouti pup in 200 pups born). We recommend that you breed your chimeras to C57BL/6 partners. Pups produced from sperm derived from ES cells will have agouti coats, half of these animals should carry the targeted gene. Pups that are produced from sperm derived from the C57BL/6 host embryo will have black coats.

1. Discard female chimeras unless you have high contribution female chimeras and a distorted sex ratio favoring females (many female chimeras and few male chimeras). You probably injected a X:O cell line. The females may very will give germline transmission.

2. Breed as many males as you can, we have observed germline transmission from males with small quantities of agouti fur.

3. Breed males aggressively from the beginning ? rotate 2 females through the male’s cage every two weeks. This will require 3 cages of 2 females in addition to the male’s breeding cage. Don’t put the male chimera into a cage occupied by females, they may attack him.

5. If the chimeras don’t produce pups by after 8 weeks of breeding and rotating females, they are infertile and should be discarded. If males produce 6 or more litters without transmitting then they are not likely to go germline and should be discarded.


Record Keeping:

1. Establish a Pedigree for each founder animal or offspring that is mated.

2. Have customized cage cards printed for mating cages and weaning cages. Mating cage cards should provide an instant summary of the activity in the cage, and serve as permanent records. The weaning card allows rapid location of mice according to line and ear tag.

3. Use a customized log sheet to record ear tag number, sex, coat color, tail DNA log number, and genotype for each mouse that is tagged and tailed. Keep the log sheets with the pedigree.

4. Together, the pedigree, mating cards, and log sheets should provide the following information for each mouse: birth date, sex, ear tag number, coat color, DNA log number, mating cage number, ear tag numbers of mother and father, source of mother and father, genotype, date mated, date euthanized, reason for euthanasia (e.g. tissue analysis), generation (how many times the line has been mated with C57BL/6).

Mouse Strain Rescue

Periodically we receive requests to help rescue mouse strains that are no longer breeding. Often the mice are beyond their preak reproductive years. Here are some of the approaches that can be used to “rescue” old strains.

1. Use C57BL/6 F1 mice to set up new breeding cages (see mouse breeding suggestions above). Most C57BL/6 F1 hybrids have good reproductive performance. We prefer (C57BL/6J X DBA/2J)F1 animals. Outbred CD-1 and ICR mice can be used, but introduce more genetic complextiy to the line.

2. Follow the breeding females for copulation plugs. Females enter estrus every 3 days or so. Mate the animals on Monday and follow them for plugs. If they haven’t plugged by Friday, keep following them over the weekend until they plug or unmate then and re-mate on Monday. Alternatively, the Transgenic Core can mate superovulated F1 female mice to your male(s) in a rederivation procedure. Eggs are collected from the females and fertilization is determined under the microscope. Fertilized eggs are transferred to pseudopregnant females to obtain pups. If the male doesn’t generate fertilized eggs (no plugs or plugs without fertilization then the next step is in vitro fertilization (see #3).

3. If the F1 females plug but don’t become pregnant then the males you are using to rescue the line are subfertile, by definition. We can use euthanize the male(s), collect sperm and perform an in vitro fertilization (IVF) procedure. The Transgenic Core routinely does IVF with excellent results. Once we rescued a line from the last male even though it died two days before the IVF. In this case, the dead male was stored in a sealed plastic bag (to prevent dessication) in the refrigerator until the IVF. Another time we rescued a mouse strain from a single spontaneous male mutant with ectopic caudal limbs in the place of a penis (polypodia mutation – Ppd gene on the X chromosome).

4. If the F1 males don’t plug the females then females are most likely anovulatory. In this case you can send the old females to the Jackson Laboratory for ovarian transplantation into a immunocompromised female (to prevent graft rejection) and you may be able to recover the line this way. The Transgenic Core does not offer this service.

5. If you took the precaution of cryopreserving the line as frozen eggs, the Core can thaw out the eggs and recover the line. If sperm were cryopreserved we may or may not be able to recover the line by IVF or ICSI, depending on the genetic background of the sperm. We recommend that frozen sperm be tested for successful IVF before a line is discontinued.

6. If the line was derived from an ES cell clone, it can be re-injected to generate a new set of germline chimeras.

7. If the line is available from another laboratory you can import them.

Transgene Transmission Patterns:

Three transgene (Tg) transmission patterns occur in transgenic founder animals. Most founder animals transmit their Tg to 50% of their offspring. About 10% to 20% of founders are mosaic for the Tg due to late Tg integration during embryogenesis. A variable proportion of founders (5% to 30%) have more than one Tg integration site. Transmission to 30% or less of offspring is a sign that the founder’s germ cells are mosaic for the Tg. In these cases it is advisable to verify the transgenic status of the founder and to breed the mouse as efficiently as possible. Founders with more than one Tg integration may transmit the Tg to 80% or more of their offspring. Different insertion sites usually segregate independently. Southern blot analysis of these offspring is used to group mice have according to insertion site. This simplifies analysis of transgene expression since expression patterns associated each independent insertion are isolated from each other. Offspring of transgenic founders transmit the Tg as normal Mendelian gene, regardless of whether the founder was mosaic or multi-integrant.

Gene Targeting Transmission Patterns:

Two transmission patterns occur in embryonic stem (ES) cell-mouse chimeras. Either they transmit or they don’t. The transmission ratios we have observed vary from 100% to 0.5%. Infertile male chimeras may also result. Since the ES cells are XY only male chimeras should be bred. We recommend that you take a male chimera through six litters before deciding to give up on it. If a chimera has a high transmission ratio, you should consider mating it directly with females from the 129 mouse strain that matches the origin of the ES cell line to obtain your targeted gene mutation on an inbred background.

Hemizygotes or Homozygotes?

We recommend that transgenic lines be maintained as hemizygotes. The primary disadvantage of maintaining lines as hemizygotes is that all offspring need to be genotyped to determine which are transgenic. Establishment of homozygous transgenic lines is is costly and entails additional breeding to produce the first homozygotes. Additional test breeding is needed to ensure homozygosity. Additional time is required to establish a homozygous line from a mosaic founder with limited transgene transmission. Extra breeding will be necessary to establish homozygous lines for each integration site in founders with multiple integrations. If this is not done carefully, animals with combinations of different integration sites will result. This will complicate interpretation of experimental results since different integration sites can have different transgene expression patterns. About 5% of transgene integration events interrupt an endogenous gene important for normal development (Meisler MH. 1992. Trends Genet. 8:341). Affected mice may display a phenotype unassociated with the transgene which may obscure the effects of transgene expression. Unequal crossing-over between transgene arrays in homozygotes may result in transgene rearrangement which will affect expression. This kind of instability occurs less often in hemizygotes. Many other difficulties can be avoided by maintaining hemizygous transgenic lines.


Table 1. Some Vital statistics of the House Mouse, Mus Musculus


number of chromosomes 40
diploid DNA content about 6 pg per cell
3X109 bp per haploid genome
recombination units 1600 centimorgans (2000 Kb/cM)
approximate number of genes§ 0.5 – 1.0 X 105
percent of genome as five families
of highly repeated DNA sequences
(B1, B2, R, MIF-1, and EC1)¥ 8 – 10%

Reproductive Biology

gestation time 19 – 21 days
age at weaning 3 weeks
age at sexual maturity 6 – 8 weeks
approximate weight birth 1g
weaning 8 – 12g
adult 30 – 40g (male > female)
lifespan in laboratory 1.5 – 2.5 years
average litter sized 6 – 8
total number of litters per
breeding female 4 – 8
useful breeding life of females 6 – 8 months
useful breeding life of males 18 – 24 months


§McKusick, V.A. and F.H. Ruddle. 1977. The status of the gene map of the human chromosomes. Science 196:390-405.
¥Bennett, K.L., R.E. Hill, D.F. Pietras, M. Woodworth-Gutai, C. Kane-Kass, J.M. Houston, J.K. Heath, and N.D. Hastie. 1984. Most highly repeated dispersed DNA families in the mouse genome. Mol. Cell. Biol. 4: 1561-1571.
Ä Parameters such as gestation time, weight, lifespan, etc., vary between the different inbred strains.
?Litter size depends on the number of eggs liberated at ovulation, the male’s sperm count, and the rate of prenatal mortality. These may vary with age of mice, parity, and environmental conditions (e.g. diet, stress, presence of strange male) and with strain (reflecting genetic factors such as efficiency of placentation). Prenatal mortality in inbred strains can be around 10-20%.



Sources of Additional Information

Table 1 is adapted from Manipulating the Mouse Embryo: A Laboratory Manual, by B. Hogan, R. Beddington, F. Constantini, and E. Lacey, published by Cold Spring Harbor Laboratory. This book has an excellent bibliography for information about mouse strains and breeding.

An excellent source is the second edition of Biology of the Laboratory Mouse, by the Staff of the Jackson Laboratory, published by Blakiston Division of McGraw-Hill.

Additional information on mouse husbandry can be found in:

Some investigators will need to know the alleles at various polymorphic loci carried by the transgenic founders. This information can be found by looking up the genotypes of the mice used to obtain fertilized eggs for microinjection in:

Genetic Variants and Strains of the Laboratory Mouse, eds. M.F. Lyon and A.G. Searle, Chapter 17; “Strain Distribution of Polymorphic Variants” by T.H. Roderick and J.H. Guidi, published by the Oxford University Press.

Other Books and Review Articles