FAQ

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.

The rule of thumb is that the more closely the strains to be separated are related, the more difficult it is to use SNPs. They can usually only be used effectively when there is backcrossing or mixing between different inbred strains, e.g. 129 and C57BL/6. However, if, for example, a mixture of different substrains of C57BL/6 is to be corrected, there are only a few markers that can be used to distinguish between donor and recipient, even when very large SNP panels are used.

This restriction does not apply to the STR markers used by GVG. Their selection was specifically based on the requirement to be able to differentiate between very closely related substrains of an inbred strain. This is not only true for substrains of C57BL/6, but also for substrains of other inbred strains, such as 129, BALB/c etc.

A particular challenge for successful backcrossing is always the chromosome on which the target is located (linked chromosome). At least two independent crossing over events are required in the DNA region around the target, one upstream and one downstream of it. The crossing over should also occur as close as possible to the target. Reality shows that such events occur relatively rarely. However, since the Speed Congenics concept calls for only 5-6 backcrossing generations, focusing on the whole-genome value, it is predictable that by generation N5, substantial amounts of donor DNA (30% - 40% or more) will still have been retained on the linked chromosome. This is a very large cluster of undesired donor DNA around the target, which then cannot be further reduced!

For this reason, in each backcross generation we conduct a targeted search for individuals in which a crossing over has occurred as close as possible to the target. If, for example, an animal with a crossing over on one side of the target is already found in the N2 generation, there are chances for the detection of a second crossing over on the opposite side of the target for three subsequent backcross generations. In parallel, there is also a steady improvement from generation to generation for the other chromosomes.

In the case of generations N4 and N5, the situation for the linked chromosome is often already satisfactorily resolved with this strategy. Here, the overall "best" is then searched for among the offspring.

For the successful implementation of such a screening strategy it is necessary that the customer provides the following information: Specify the chromosome and the approximate position of the target on the chromosome (in MBp is sufficient). In addition, knowledge of the exact name of the recipient strain is required. When using C57BL/6, it is not sufficient to simply refer to the suffix "J" or "N". Both "J" and "N" have multiple substrains, which in turn are genetically distinct (JBomTac, JOlaHsd, JCrl, JRj, JRccHsd, NTac, NCrl, NHsd, NRj).

Furthermore, it is very helpful if a DNA sample of the F1 generation is provided. This can be used to initially "calibrate" the overall system for genotyping, i.e. to search for informative (= heterozygous) markers that can be used for the particular project.

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.

Experience has shown that there are several problems with the simultaneous backcrossing of two or more different targets in one line. First, one needs a very large number of offspring to identify among them those that possess both targets at the same time. This can usually only be achieved by a much larger number of matings, or by mating several times in succession.

On the other hand, it shows that desired crossing over events can usually only be detected for one of the two targets. On the other hand, another animal shows a crossing over for the second target. This ultimately leads to target-specific recommendations for the continuation of the project and to a bloating of the breeding from generation to generation. Both situations ultimately lead to a considerable delay of the entire backcrossing project and to a very large number of individuals that are not needed further (breeding surplus).

We have made the experience that the optimal solution is to divide such projects from the beginning, to cross back each target separately in parallel and to bring them together again only afterwards. In addition to the time factor, this is also in the interest of 3Rs, since only a manageable number of breeding pairs need to be used for each subproject and, in terms of the total number, considerably fewer animals are required for successful completion of the project. An additional advantage of this strategy is that each target is available as a single-mutant mouse line and thus, if necessary, the study can be extended to include comparative tests between single and double mutants in the identical recipient strain.

Speed congenics only lives up to the name "speed" if the best of a limited number of candidates can be selected per generation for further breeding. This number is ideally between 5 and 8. Mathematically, every fourth animal among the offspring is male and at the same time target-positive. The total number of offspring needed is therefore between 20 (4 x 5) and 32 (4 x 8).

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.

If the target is located on the X chromosome, a crossing over event must have taken place on both sides of the target for the exchange of the donor parts by recipient DNA. However, this is only possible in females, since males pass on their single X chromosome unchanged and thus do not contribute to any progress in backcrossing. The rule is that only after a satisfactory situation on the X chromosome (crossing over on both sides of the target) it is possible to switch to the use of male offspring. This is the case at the earliest from backcross generation N3, mostly later.

It is recommended to test as large a number of target-positive females of the N2 generation as possible, the more the better. This can be achieved by mating F1 females with males of the recipient strain (only this constellation!) in parallel with a larger number of matings. From the technical point of view this is completely uncritical, because all females of the F1 generation are identical among themselves with regard to the proportion of donor/recipient (50:50).

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.

During backcrossing, the fixation of both sex chromosomes of the recipient strain must be done. If available, only the mating of a female donor animal with a male recipient is generally recommended. This ensures that all male F1 offspring already possess the correct Y chromosome of the recipient. The X chromosome, on the other hand, is still from the donor. With the recommended strategy that only male offspring are used for further breeding, all males of the N2 generation then already also possess the X chromosome of the recipient strain. Both sex chromosomes then exclusively possess DNA of the recipient.

Mistakes, on the other hand, are often made with the Y chromosome when the donor animal is male and no female donor animal is available. In this case, the Y chromosome of the recipient must first be crossed in. This is very often forgotten. A "wrong" Y chromosome may have significant effects on the phenotype even in female offspring due to epigenetic effects, which may confound the experimental results of a study. It is essential to fix the Y chromosome with the N2 generation by starting several breeding pairs in parallel for this purpose, with female target-positive animals of the F1 generation and male recipient animals. All male individuals of the resulting N2 generation thus exclusively possess the correct Y chromosome. Only male animals are used to perform the backcross from the N2 generation onwards. Thus, the correct Y chromosome of the recipient strain is retained in all offspring. If this time of fixing the Y-chromosome is missed, this can be done later only with considerable loss of speed in backcrossing, because then the one best female has to be mated with a recipient male and among the 6-8 offspring of the litter only very few target-positive males can be expected (see point: Why are exclusively male offspring used).

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.

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