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)

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). 

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


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Genome


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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%


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Reproductive Biology


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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


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§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 

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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:
Handbook on Genetically Standardized JAX Mice, 1997. by the Staff of the Jackson Laboratory, available from The Jackson Laboratory.
Systematic Approach to Evaluation of Mouse Mutations. 2000. Sundberg, JP and Boggess D, eds. CRC Press, LLC, Boca Raton, Florida.
Mouse Genetics and Transgenics: A Practical Approach. 2000. Jackson, IJ, and Abbott, CM, eds. Oxford University Press, New York.
Mouse Genetics: Concepts and Applications. 1995. Silver, LM. Oxford University Press, New York. - This Book is Available Online.
The NIH Guide for the Care and Use of Laboratory Animals is available online.

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

comments to: tsaunder@umich.edu