Frequently asked questions from our customers
Questions about Speed Congenics
Speed Congenics, also referred to as "Marker assisted selection protocol" (MASP), is a method for the accelerated generation of congenic mouse lines. These are lines in which the smallest possible, defined DNA segment containing a modified gene (hereafter referred to as the target) has been transferred from one donor strain to another strain (recipient). The entire remainder of the genome of the congenic mouse line consists solely of recipient DNA. This transfer is achieved by a stepwise backcrossing, in which the offspring of a backcross generation are always mated again with animals of the recipient strain. From generation to generation, the genomic proportion of donor DNA thus decreases continuously.
Within each backcross generation, those individuals possessing the target are first identified. These are then genotyped using 200-250 informative STR markers (short tandem repeats, microsatellites), which are present at regular intervals across all chromosomes and can distinguish between donor and recipient. For the generation of the next backcross generation, the animal is used which already has the highest genomic proportion of the recipient strain among the offspring. This allows the total duration of backcrossing to be reduced from the original 10 generations in the classical method to 5-6 generations. This leads to a time saving of 1-1.5 years.
If an existing mixed genetic background of a mouse line is to be traced back to a defined substrain of an inbred strain of the mouse, the analogous method is used as practiced for speed congenics.
There are donor animals for which the integration site of the target is not known. Using GVG's technology platform, it may be possible to identify the affected chromosome and determine the approximate position of the target on the chromosome from the availability of genotyping results from approximately 7-10 individuals. Based on this, it is then possible to proceed with the described strategy of "best crossing over" in a targeted manner. If the linked chromosome cannot be determined, the genome-wide most advanced "best" is searched for.
If one would use the one best female for mating, such a number of offspring can only be achieved by 3 successive matings/pregnancies. A male, on the other hand, can be mated with 3 females in parallel in a short period of time without any problems. One gets with one male the same number of offspring within one generation, in a threefold shorter period.
The generation of the N2 generation can be regarded as a special feature/exception. In this early stage of backcrossing, a first attempt is made to identify an animal in which a crossing over event has already occurred as close as possible to the target. The more male (target-positive) N2 animals can be tested, the better the chances are. This can be achieved by setting a larger number of breeding pairs in parallel for the mating of F1 females with males of the recipient strain. This is possible from the point of view of genetics, since all animals of the F1 generation are identical among themselves with regard to the proportion of donor/recipient. For the subsequent backcross generations, on the other hand, only the best animal recommended by us on the basis of the genotyping results should then be used for further matings.
The first step in backcrossing is to mate a target-positive donor with an animal of the recipient strain. All animals of the resulting F1 generation contain 50% DNA of the donor and 50% DNA of the recipient. If the donor was homozygous with respect to the target to be transferred, all animals of the F1 generation contain the target and therefore do not need to be analyzed. If, on the other hand, the target is present in heterozygous form in the donor, those animals that also possess the target must first be determined among the F1 offspring. In both cases, genome-wide genotyping is not yet necessary.
An F1 DNA sample should be provided to the GVG to identify all informative, (=heterozygous) STR markers.
The letter "F" is used to designate successive generations in inbred strains, which are produced exclusively by brother-sister matings. In backcrossing, however, the mating scheme is different, here always an offspring of the respective backcross generation is mated with an animal of the recipient. To indicate this difference, the letter "N" is assigned in Speed congenics. After successful completion of such a project, a switch is then made to a brother-sister mating. From this point on, the number of generations of the breeding of the new line is marked by the letter "F", combined with the respective number of generations.