Backcross vs Testcross difference: Research on mitochondrial genes

  • Thread starter Beth N
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Summary
I am a college student trying to interpret a biology research paper. The paper is studying the link between mitochondrial gene mutation and changes in gut microbiota composition, using the mice model.
My question is all in the context of this paper. I have problems understanding a definition and a method.
Sina, C. et al. Mitochondrial gene polymorphism is associated with gut microbial communities in mice.
Sci. Rep. 7, 1–9 (2017).

This is a segment of the introduction: "
Since common inbred mouse strains demonstrate unique mitochondrial
genomes
and the mitochondria are strictly maternally inherited, we systematically generated conplastic
strains carrying mostly single mutations in mtDNA with a C57BL/6J nuclear background by repeatedly back
crossing
female mitochondrial-donor strains with male C57BL/6J mice over 12 generations. This unique
resource allowed us to investigate the impact of defined mtDNA mutations on gut microbiota composition in
the absence of confounding variation in the nuclear genome."

I don't understand the method by which they generate " complastic mouse strains" and "backcrossing".
Does backcrossing mean making a breed between an offspring and its parents or another indiividivual with the same genotype as its parent?
And does backcrossing the same thing as testcrossing?

What I understand:
- The purpose of backcrossing is to generate mice strains that only differ in their mitochondrial gene, but not their nucleic gene, so that any difference in the gut microbiota composition is not confounded by difference in the nucleic genes of the mice.

What I don't understand:

I think I'm thinking about how they actually carry out the procedure completely wrong :(. Here's what I've got:


Parents: Male C57BL/6J x Female with same nucleic gene but mutated mitochondrial gene
Generation F1: An offspring male C57BL/6J (same nucleic gene as its father) * any female offspring (<-- could it be a female offspring, or a female unrealted mouse with a different mitochondiral mutation?)
Generation F2: An offspring male C57BL/6J * any female offspring
Repeat for 12 generations.

Any clarifiication would be greatly appreciated!
 

BillTre

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Overview:
Its sounds like you understand the difference between the nuclear genome and the multiple copies of the mitochondrial genome in cells.
Many different slightly different versions of the mitochondrial genome can be inherited from the mother. The father does not contribute to their offsprings's collection of mitochondrial genomes.
The nuclear genome (C57BL/6J)is well defined can be genetically manipulated using standard Mendelian crossing methods.
Since you are starting with a 57BL/6J background and all the crosses are made on the 57BL/6J background. It is a reasonable to assume that there is no difference between the different 57BL/6J backgrounds (unless otherwise stated in the paper). Therefore making crosses between the 57BL/6J background mice should have no effect on the genetic background of the mice. On the other hand, there maybe some variability in the 57BL/6J genetic backgorund they are using (this can happen for a variety of reasons). Backcrossing to a male ancestor (whch could be prolonged through 12 generations by using frozen sperm) would be a way of reducing the 57BL/6J background variability.
Mitochondrial genome manipulations can only done by controlling the female side of the crosses.
Multiple mitochondria (and their genomes) are inherited from the mother as a subset of the different versions of mitochondrial genomes present in her germ cells. This can be a diverse set of genomes with genetic differences.
To make lines of mice with identical mitochondrial genomes, they used 12 generations of backcrosses.

Conplastic strains would (presumably) be strains of mice with identical mitochondrial genomes).
Once a female mouse is generated with only one kind of mitochondrial genome, then it could be expanded in to a line of identical mice that could be tested vs. other lines of mice.

Test crosses
are crosses set-up to detect a genetic condition that is not easily determined in an animal. An example would be to determine if some animal were carrying a recessive mutation as a heterozygote. This is most easily done by crossing the animal to a known carrier for the recessive gene in question. If the first animal is a carrier, then the cross should have ~1/4 of the offspring as showing the recessive trait (however, any recessives would be taken as a positive result). This is an analytic method.

Backcrosses are crosses between offspring and parent (usually). They could be used analytically but usually for producing some particular genetic result (therefore a production method).
In this case, they are want to reduce the number of different kinds of mitochondrial genomes in a line to one.
Back crossing a parent (or between siblings) is a common method for generating a line of mice, all with the same nuclear genome. 10-12 generations of crosses is often considered enough to generate isogenic lines. If they were backcrossing to a single male, then the 57BL/6J nuclear genome they carry would approach the 57BL/6J genetic background which the single male carried. Different female carried mitochondrial genomes with different genetic varients could be crossed in while maintaining 57BL/6J background. Crossing to females of the 57BL/6J would preserve most of the nuclear genetic background. Crossing back to the same original male would then tend to "washout" nuclear genes from the female over teh following generations.

WRT the mitochondrial genome, since each new offspring might (by chance) not inherit all of the variability of the parent, the offspring could be sorted through (presumably using molecular genetic techniques) to find those offspring carrying the least variability in their mitochondrial genomes.
By repeatedly finding and breeding mice carrying fewer variants of the mitochondrial genomes in each generation, they could eventually generate mice with a single version of the mitochondrial genome. These individual mice could then be used to start a line for their experiments (or tests). If this were repeated several times in parallel, then several lines differing only in the kinds of mutations in the mitochondrial genome will be generated. Each different line being conplastic. This might have already been done since they mantion mitochondrial donor lines. However, since mitochondrial genes are reproduced more times than the genes of their host cells and have a higher mutation rate, any previously generated lines could redevelop new mutations and therefore variability in their mitochondrial genomes.

I'm guessing that backcrosses were used in this aspect of the line generation to eliminate residual variation in the 57BL/6J genetic background while a collection of lines with different mitochondrial genomes on the 57BL/6J background. Sibling crosses would all inherit closely related mitochondrial genomes since they would all have the same mother, but if there were variability in the 57BL/6J background they had available to use.

Since they mention mitochondrial donor strains, they may already have strains of mice with well defined mitochondrial genomes which they used to cross into the lines they wanted to make.

If your interpretation:
Parents: Male C57BL/6J x Female with same nucleic gene but mutated mitochondrial gene
Generation F1: An offspring male C57BL/6J (same nucleic gene as its father) * any female offspring (<-- could it be a female offspring, or a female unrelated mouse with a different mitochondrial mutation?)
Generation F2: An offspring male C57BL/6J * any female offspring
Repeat for 12 generations.
is correct, then they used the backcrosses only to initially generate the lines, after which they were further inbred using sibling crosses.
The backcross would be to add mitochondrial variants, while the further breeding would be to make the lines more 57BL/6J isogenic.
 

Ygggdrasil

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Insights Author
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In the paper, they say that the generation of the conplastic strains is described here: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2612955/

Essentially, they found that different mouse strains have very similar mitochondrial genomes aside from a few mutations. They wanted to know the effects of those mutations, but to do that they can't just directly compare two different strains of mice as these mice would differ both in their mitochondrial and nuclear genomes.

To isolate the effects of the mitochondrial genome, they wanted to basically put all of the different mitochondrial genomes into the C57BL/6J background. To do this, they mated a female from the mouse strain of interest with a male C57BL/6J mouse. This produces a progeny containing the mitochondrial genome of interest and about a 50% C57BL/6J nuclear genome. They then take a female progeny and mate with another male C57BL/6J. Their progeny will now have 75% C57BL/6J nuclear genome. They repeat this process for a total of 12 matings to produce a mouse retaining the mitochondrial DNA from the original mouse strain, but almost no nuclear DNA from that original strain (only about 1/2^12 or about 0.02% nuclear DNA would remain from the original strain).
 
37
3
Overview:
Its sounds like you understand the difference between the nuclear genome and the multiple copies of the mitochondrial genome in cells.
Many different slightly different versions of the mitochondrial genome can be inherited from the mother. The father does not contribute to their offsprings's collection of mitochondrial genomes.
The nuclear genome (C57BL/6J)is well defined can be genetically manipulated using standard Mendelian crossing methods.
Since you are starting with a 57BL/6J background and all the crosses are made on the 57BL/6J background. It is a reasonable to assume that there is no difference between the different 57BL/6J backgrounds (unless otherwise stated in the paper). Therefore making crosses between the 57BL/6J background mice should have no effect on the genetic background of the mice. On the other hand, there maybe some variability in the 57BL/6J genetic backgorund they are using (this can happen for a variety of reasons). Backcrossing to a male ancestor (whch could be prolonged through 12 generations by using frozen sperm) would be a way of reducing the 57BL/6J background variability.
Mitochondrial genome manipulations can only done by controlling the female side of the crosses.
Multiple mitochondria (and their genomes) are inherited from the mother as a subset of the different versions of mitochondrial genomes present in her germ cells. This can be a diverse set of genomes with genetic differences.
To make lines of mice with identical mitochondrial genomes, they used 12 generations of backcrosses.

Conplastic strains would (presumably) be strains of mice with identical mitochondrial genomes).
Once a female mouse is generated with only one kind of mitochondrial genome, then it could be expanded in to a line of identical mice that could be tested vs. other lines of mice.

Test crosses are crosses set-up to detect a genetic condition that is not easily determined in an animal. An example would be to determine if some animal were carrying a recessive mutation as a heterozygote. This is most easily done by crossing the animal to a known carrier for the recessive gene in question. If the first animal is a carrier, then the cross should have ~1/4 of the offspring as showing the recessive trait (however, any recessives would be taken as a positive result). This is an analytic method.

Backcrosses are crosses between offspring and parent (usually). They could be used analytically but usually for producing some particular genetic result (therefore a production method).
In this case, they are want to reduce the number of different kinds of mitochondrial genomes in a line to one.
Back crossing a parent (or between siblings) is a common method for generating a line of mice, all with the same nuclear genome. 10-12 generations of crosses is often considered enough to generate isogenic lines. If they were backcrossing to a single male, then the 57BL/6J nuclear genome they carry would approach the 57BL/6J genetic background which the single male carried. Different female carried mitochondrial genomes with different genetic varients could be crossed in while maintaining 57BL/6J background. Crossing to females of the 57BL/6J would preserve most of the nuclear genetic background. Crossing back to the same original male would then tend to "washout" nuclear genes from the female over teh following generations.

WRT the mitochondrial genome, since each new offspring might (by chance) not inherit all of the variability of the parent, the offspring could be sorted through (presumably using molecular genetic techniques) to find those offspring carrying the least variability in their mitochondrial genomes.
By repeatedly finding and breeding mice carrying fewer variants of the mitochondrial genomes in each generation, they could eventually generate mice with a single version of the mitochondrial genome. These individual mice could then be used to start a line for their experiments (or tests). If this were repeated several times in parallel, then several lines differing only in the kinds of mutations in the mitochondrial genome will be generated. Each different line being conplastic. This might have already been done since they mantion mitochondrial donor lines. However, since mitochondrial genes are reproduced more times than the genes of their host cells and have a higher mutation rate, any previously generated lines could redevelop new mutations and therefore variability in their mitochondrial genomes.

I'm guessing that backcrosses were used in this aspect of the line generation to eliminate residual variation in the 57BL/6J genetic background while a collection of lines with different mitochondrial genomes on the 57BL/6J background. Sibling crosses would all inherit closely related mitochondrial genomes since they would all have the same mother, but if there were variability in the 57BL/6J background they had available to use.

Since they mention mitochondrial donor strains, they may already have strains of mice with well defined mitochondrial genomes which they used to cross into the lines they wanted to make.

If your interpretation:

is correct, then they used the backcrosses only to initially generate the lines, after which they were further inbred using sibling crosses.
The backcross would be to add mitochondrial variants, while the further breeding would be to make the lines more 57BL/6J isogenic.
Thank you very much!
 
37
3
In the paper, they say that the generation of the conplastic strains is described here: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2612955/

Essentially, they found that different mouse strains have very similar mitochondrial genomes aside from a few mutations. They wanted to know the effects of those mutations, but to do that they can't just directly compare two different strains of mice as these mice would differ both in their mitochondrial and nuclear genomes.

To isolate the effects of the mitochondrial genome, they wanted to basically put all of the different mitochondrial genomes into the C57BL/6J background. To do this, they mated a female from the mouse strain of interest with a male C57BL/6J mouse. This produces a progeny containing the mitochondrial genome of interest and about a 50% C57BL/6J nuclear genome. They then take a female progeny and mate with another male C57BL/6J. Their progeny will now have 75% C57BL/6J nuclear genome. They repeat this process for a total of 12 matings to produce a mouse retaining the mitochondrial DNA from the original mouse strain, but almost no nuclear DNA from that original strain (only about 1/2^12 or about 0.02% nuclear DNA would remain from the original strain).
Thank you very much!
 

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