what is the best concentration of hyg for selecting transgenic barley plants? How to distinguish the transgenic plants and non-transgenic plants? growing slowly on selection plate? or other phenotype? Some people recommended negative plants will grow slowly. But, it is also probably resulted by seed viability.
I am a little bit confuse about this.
if there are some people can answer this that will be very helpful
Kind regards
Transgene copy numbers can also be checked with Southern analysis (with radioactive- or non-radioactive-labeled hybridization probes).
Radioactive probe-- ex. with P32
Non-radioactive probe-- ex with digoxigenin (DIG).
You can also use 'progeny test' to find out homozygous transgenic lines. Take a diploid selfing species and a single transgene insertion as example. If the parental transgenic line is hemizygous/heterozygous (ex. A/-), you will have a progeny population of A/A, A/- and -/- plants derived from this parental line. In this case, some progeny plants (A/A and A/-) will survive selection and some will not (-/-). If the parental transgenic line is homozygous (ex. A/A), its progeny will all have A/A genotypes, and should all survive selection, provided that the transgene is not silenced. Transgene silencing can be due to 'position effect', 'methylation' or other factors.
There are a few methods can be used to determine the zygosity of transgenic plants.
Attached is a review paper about this topic. It summarizes almost all the methods which have been employed for transgenic plant zygosity determination.
Paper title: "Assessing zygosity in progeny of transgenic plants: current methods and perspectives".
Hii friend. As Prof. Yuan mentioned Southern blot is recognized way to confirm homozygosity. Besides that please also consult attached article, how fellow researchers used Real Time PCR to address homozygity in transgenes.
Article Quantitative real-time PCR assay to detect transgene copy nu...
Article Analysis of the copy number of exogenous genes in transgenic...
Southern blot is a standard way to assess 'transgene copy numbers', probably not 'zygosity (homo- vs. heterozygous)', at least in plant (not sure in animal field). In plants, most published Southern data are used for assessing transgene copy number.
In theory, Southern blotting can be used for detecting homozygous and heterozygous plants by comparing the intensity of the 'signals' on the X-ray film (autoradiograph) [see attachment]. The siganals should be more intense for homozygous plants (with 2 identical copies) than for heterozygous plants (with 1 copy). This can be done with a densitometer. However, the results on the autoradiograph can be ambiguous, due to conditions of probe hybridization, wash, and other steps of this method.
1. qPCR can also be used to assess transgene copy numbers as mentioned. There are plenty of reports online can be found for references.
2. Attached is a 2016 paper. Authors used iPCR, qPCR and Southern analysis for transgene copy number assessment and compared them. They found:
(1) iPCR is a robust method for transgene copy number determination, especially when the transgenic plant is with 'single-copy' transgene insertion. The detected PCR band is clear and single. However, while the insertion copy number is increased, the results become ambiguous. Prominent and faint bands can appear together. In this case, more analyses (ex. cloning and sequencing) of these bands are needed to further clarify the results.
(2) qPCR can be used for zygosity or transgene copy number determination. However, this method is based on the assumption that the 'PCR efficiency' for both 'target gene' and internal 'reference gene' is the same. But, in reality, it may not. So, sometimes qPCR results can be inaccurate (see attachment, 2015). Due to this disadvantage, other methods/technology have developed to replaced older qPCR method.
Examples of those new methods are: SA-qPCR (see attachment) and Droplet Digital™ PCR (ddPCR™) Technology
In animal, fluorescent in situ hybridization (FISH) Is an option to use for zygosity determination. I don't know whether it can be efficiently applied to plants. See the attached paper:
Title: ZyFISH: A Simple, Rapid and Reliable Zygosity Assay for Transgenic Mice
Hi :)
I work with Arabidopsis. What I do is first I grow my transgenic seeds on MS plate which includes ppt (1:1000) or hyg (1:2000) resistance (based on the vector I had used to make my transgenic lines), this leads to a lesser number of non-transformed seeds germination, then I transplant the seedlings to soil and when they're bigger, I take leaf samples for DNA extraction and PCR to identify the homozygotes between them. I only collect seeds from those homozygote ones then.
I hope this can help.
Follow up question, Why there is a need to prepare fluorescent probes for q-PCR to find out homozygous transgenic lines
when we can just compare the endogenous reference with transgene??
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