FAQ's
What are chimeras?
Typically, chimeras are constructed to generate mice from gene-targeted or gene trapped ES cell lines. ES cells injected into host embryos give rise to mosaic mice known as chimeras.
Male ES cells are injected into unsexed
blastocysts. If the host embryo is female and the male ES cells make germ
cells, the chimera will often be a fertile male. Host embryos from a strain which won't compete too strongly with the ES cells are used, and the two components (ES and host embryo) are genetically marked with coat color genes so that the contribution of the ES cells to the animal can be determined easily. If the proportion of ES cell descendents in the coat of the animal is high, the probability that ES cells are represented in gametes is also high, since ES cells mix thoroughly with host cells early in embryogenesis. The coat color marking system also makes it possible to determine, with appropriate crosses, if the offspring of the chimeras are from the ES cell component or host embryo component. 129 ES cells give rise to brown coat
color because they are A/A (wild type Agouti),
and the C57BL/6J host embryos give rise to black coat color because they are a/a (recessive nonagouti). The ES cells are from the 129 strain of mice; the host embryos are from the C57BL/6J strain of mice. If these chimeras are bred to a/a non-agouti mice (for example C57BL/6J, then any brown offspring (A/a) must have arisen from ES cell-derived gametes, and 50% of the brown offspring are expected to carry the knockout allele. Alternatively, the transmission of the mutation can be assayed directly by PCR or Southern blot assays of tissues from the offspring.
If the ES cells are from the C57BL/6J strain (a/a, Tyr/Tyr and black), the host embryo will be albino C57BL/6J (a/a, Tyrc-2J/Tyrc2-J and white). Breeding such chimeras to albino C57BL/6J can generate black offspring (a/a, Tyr/Tyrc-2J) from the ES cell component, 50% of which should carry the mutation, and white offspring (a/a, Tyrc-2J/Tyrc-2J) from the host embryo component.
Chimeras for other uses
We also make aggregation chimeras and diploid<==>tetraploid chimeras. Two preimplantation embryos from two different strains of mice are combined to form an aggregation chimera. This technology can be used to generate genetic mosaic embryos for analysis of autonomy and nonautonomy of gene action and other experimental goals. In diploid<==>tetraploid chimeras, the tetraploid component largely gives rise to much of the embryo-derived placenta and the diploid component to the embryo. This type of chimera can be used to determine if a gene is required in placenta or embryo.
Why are my mice funny colors?
Some ES cell lines are heterozygous for coat color alleles that only reveal themselves in subsequent generations. The R1 129 ES cells we use are heterozygous at albino locus, carrying one copy of chinchilla allele of albino (cch, also designated Tyrc-ch) and are heterozygous at the pink-eyed dilution locus (p). Breeding offspring descended from R1 ES cells to each other can give rise to mice which are black, different shades of gray, different shades of brown, or yellow, depending on the assortment of coat color alleles.
What can be done to ensure germline transmission?
Germline transmission is maximized with ES cells
that have spent a minimum amount of time in culture, that have a normal complement of chromosomes, and that are free of yeast, bacteria and mycoplasma. Target a cell line that you know is transmitted under your conditions at your institution. Use a parental cell line that is at the lowest passage number available (preferably less than passage number 16). Minimize the time that the targeted line is cultured. Verify that targeted cell lines have the normal number
of chromosomes. Test your cell line for mycoplasma. Some cell lines will not transmit even when all these parameters are in your favor. Even under optimal conditions, only two-thirds of targeted ES cell lines will be transmitted through the germ line. Therefore, start with at least three independent targeted cell lines to ensure transmission.
I have weak chimeras/female chimeras. Will they transmit the knockout?
Both weak chimeras and female chimeras can transmit mutant alleles, although infrequently. Only about 10% of female chimeras transmit the ES cell genome, when they do so it is only in the first few litters, and many of their male offspring have sex chromosome aneuploidies which render them infertile (Bronson et al., 1995 PNAS 92, 3120 .pdf).
Why did my strongest chimeras die?
Mortality can increase with the proportion of the animal that comes from ES cells, even for the best cell lines. This mortality is due largely to epigenetic aberrations which arose in culture. The epigenetic aberrations are thought to be corrected by subsequent breeding of surviving mice, and would segregate randomly away from the targeted locus, even if they were not.
What strain of mice do I breed my mutants to?
To minimize genetic variability as quickly as possible breed the chimeras to the same background as the ES cells. If the ES cells were 129, breed the chimeras to 129 mice and your mutation will be completely on the 129 inbred background in one step. The most common inbred background for genetic studies is the C57BL/6 strain. If your ES cells are C57BL/6, cross your chimeras to C57BL/6J mice. If you wish to change the background, the standard is to do 9 serial crosses to the inbred strain of choice. The rationale for the 9 generations is explained here by Lee Silver.
For maximum reproduction, cross your mice onto an outbred strain like CD1. CD1 mice were derived from Swiss mice, have a high degree of genetic variation and were selected for robust reproduction.
Investigators need an IACUC protocol from their institution.
WHAT YOU WILL PROVIDE PART I
An account number.
A mycoplasma-free ES cell line with 40 chromosomes.
WHAT WE WILL DO
We will inject approximately 50 blastocysts per ES cell line. This should result in 10 mice born. Three of the mice are expected to be strong male chimeras. We have a proven record of success.
We will house the mice until weaning (three weeks after birth).
WHAT YOU WILL PROVIDE PART II
You will house the weaned mice.
COST
$2,500 per cell line. An additional minimum of $200 for animal transport will be charged for non-CWRU orders.
ACKNOWLEDGEMENTS
We ask that you acknowledge contributions of the Case Transgenic and Targeting Facility in seminars and publications.