F1 generation - (Jul/23/2008 )
From my positive founder transgenic mouse, when i mated with a WT female, i got 9 pups, out of which only 3 are transgenic . Why ?
I have just started working with transgenics, so am sure I am missing some basic concepts, any reading material or link would also be helpful.
thanks!
maybe your transgenic parent was a heterozygote? If is, then you would expected only half your baby mice to be transgenic 0.5*9=4.5 which is quite close to 3.
Thanks Perneseblue, can the heterzygosity be determined and if yes how?
Can it be because of difference in copy number, thats another thing which my lab mates are suggesting to determine in my transgenic pups. if you are familiar , can you please let me know how to determine the copy number ?
Thanks for your help.
I guess it depends on how much information you know about your gene and the mutant gene. But strictly speaking this isn't my field of experties.
Do you know the nature of the transgenic parent?
Heterzygosity can be determine by tetraARM-PCR if you know which bp are different. If you know what bp is different you can design the appropriate primer. If you don't know which bp are different, I guess you could PCR amply the gene and sequence it. If you examine the sequence read, any heterozygocity should appear as a double peak. This method of course will depend on how good you are at getting good sequence reads.
As for gene copy number, if you know where the gene sequence, you can run a southern blot. Difference in copy number will be seen as a difference in size or band intensity (assuming equal loading of genomic DNA). Quantitative PCR is also another option for copy number detection
Do you know the nature of the transgenic parent?
Heterzygosity can be determine by tetraARM-PCR if you know which bp are different. If you know what bp is different you can design the appropriate primer. If you don't know which bp are different, I guess you could PCR amply the gene and sequence it. If you examine the sequence read, any heterozygocity should appear as a double peak. This method of course will depend on how good you are at getting good sequence reads.
As for gene copy number, if you know where the gene sequence, you can run a southern blot. Difference in copy number will be seen as a difference in size or band intensity (assuming equal loading of genomic DNA). Quantitative PCR is also another option for copy number detection
Thanks perneseblue.
No, I do not know the nature of transgenic parent, i think sequencing will be the best way to go for me. Can you explain why will a double peak is expected in heterozygous nature?
I know the cDNA sequence of the gene, but do not know where it is located in the genome. How can it found ? This is my first time working with transgenic, so even i am not experienced.
Thanks for your suggestion.
well, lets assume that at at position 123 of your gene, the mutant gene has an A, while the wild type gene has a C.
A heterozygote will have both the wildtype gene and the mutant gene. If you were to PCR amplify a heterozygote using primers, you will produce two PCR products. The mutant product and the normal product. If you where then to sequence this mix, you will produce a sequence read of both products. Thus where the mutant and wild type differ you will have two peaks overlapping. Ie at position 123, you will see a peak for A overlapping a peak for C.
Is there anyway to find out about the transgenic parent? Like how it was made? Transposon knock out? chemical mutagenesis? Any information at all? Is it possible to communicate with the person who made this transgenic mouse? You need to find out how this mouse was made.
As for the gene, if you know the cDNA sequence, you should be able to find out the gene (given that the mouse genome has been sequenced- blast search). And from there you can tell approximately where the gene is in the genome, as different backgrounds of different mouse strains do lead to some variation in the exact location of the gene.
And since you can find the gene, you can find the exons that compose the cDNA sequence. Knowing the exons, you can then design southern blot probes.
ah... and I am being a little dense, the most basic means to determine if an organism is homozygote or heterozygote is to see if it breeds true. Do you have a male and female of the transgenic?
A heterozygote will have both the wildtype gene and the mutant gene. If you were to PCR amplify a heterozygote using primers, you will produce two PCR products. The mutant product and the normal product. If you where then to sequence this mix, you will produce a sequence read of both products. Thus where the mutant and wild type differ you will have two peaks overlapping. Ie at position 123, you will see a peak for A overlapping a peak for C.
Is there anyway to find out about the transgenic parent? Like how it was made? Transposon knock out? chemical mutagenesis? Any information at all? Is it possible to communicate with the person who made this transgenic mouse? You need to find out how this mouse was made.
As for the gene, if you know the cDNA sequence, you should be able to find out the gene (given that the mouse genome has been sequenced- blast search). And from there you can tell approximately where the gene is in the genome, as different backgrounds of different mouse strains do lead to some variation in the exact location of the gene.
And since you can find the gene, you can find the exons that compose the cDNA sequence. Knowing the exons, you can then design southern blot probes.
Thanks perneseblue, for the detailed explanation.
The transgenic was made by a point-mutagenesis in cDNA which was alraedy used to make a different transgenic mouse. after removing the extra part of the vector and purifying it was microinjected and then transferred to a pseudopregnant mother. The founders were genotyped and few of the founders are the parents.
Can you please tell me briefly how to search for a gene in mouse genome, so that i can see the exons and introns. the accession number of the gene is NM_005208.
ah... and I am being a little dense, the most basic means to determine if an organism is homozygote or heterozygote is to see if it breeds true. Do you have a male and female of the transgenic?
Yes, I do have a male and female transgenic pups.
F1 hybrid is a term used in genetics and selective breeding. F1 stands for Filial 1, the first filial generation seeds/plants or animal offspring resulting from a cross mating of distinctly different parental types. The offspring of distinctly different parental types produce a new, uniform variety with specific and/or desirable characteristics from either or both parents. In fish breeding, those parents frequently are two closely related fish species, while in plant and animal genetics those parents usually are two inbred lines. Mules are F1 hybrids between horse and donkey.
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