Transgene Het-Hom Genotyping Service

The Transgene Het-Hom Copy Number service is a useful tool to:

• Determine the Heterozygous or Homozygous status of randomly inserted transgenes using copy number testing

• Monitor the genetic stability of the copy number

What are the benefits of identifying the copy number in my rodent colonies?

There is growing understanding of copy number variation as a potential cause pf phenotypic variation. Mutations resulting in genetic drift is a widely recognised natural consequence of breeding multiple generations of laboratory rodents. We no longer need to wait for phenotypes to disappear, we can proactively ensure we are maintaining colonies with appropriate copy numbers. This can save time, money, and resources. Maintaining a moderate sized colony (25 cages) costs over $10,000 per year in agistment alone. Having confidence in our animal models and limiting genetic instability ensures research resources are effectively managed.

Knowledge of the transgene genotype and copy number will allow research groups to:

• Select the most appropriate breeders based on genotype e.g. Het vs Hom. Removing the guess work when trying to breed to a specific genotype
• Reducing time taken to breed to required genotypes
• Reduce the number of animals born and subsequently culled
• A tool to monitor copy number variability over time for uncharacterised strains
• A tool to investigate unexpected changes in phenotypic characteristics

How can I submit samples?

Please contact GRS to see if we have any existing assay for your transgene or if a new assay would need to be designed. We will provide existing clients with an updated Genotyping submission form. Please instruct the animal facility to collect tissue for the animals you would like tested for this service.

• Samples submitted should follow the definition of a ‘standard sample’ found in our service agreement
• Please collect two samples per animal if you have not used this service before, this will allow us to validate custom test designs
• If submitting samples for Het / Hom determination, please ensure the animal has previously been genotyped as Transgene Positive to avoid processing samples that are not carrying the Transgene. 
• If studying variability in copy number, we recommend submitting samples from an appropriate number of animals across a number of litters. 

How are the results provided?

Based on your submission form we will report the:

• Genotype status for the transgene including controls used to determine the genotype
• The copy number including statistical evidence to demonstrate how this was verified. This will allow research groups to identify changes over time.

How do we ensure quality results?

• We will run repeats
• Technical duplicates are used to ensure they match review error bars
• We use controls in all sample processing at Genetic Research Services  

What method is used to obtain the copy number?

GRS genotypes most transgenes using traditional endpoint PCR. While this method can detect the presence or absence of a transgene (Positive/Negative) it is not a quantitative method and cannot determine the number of copies of a transgene that are present in the genome. For studies or breeding programs that require determination of the copy number of the transgene (zygosity) we use a quantitative PCR method called Digital PCR.

Digital PCR is similar to endpoint PCR, except that prior to PCR amplification the reaction is divided into thousands of separate partitions. Dividing the reaction in this way means that some partitions will contain a copy of the transgene DNA and other partitions will not. The PCR reaction is then performed and the number of partitions that are positive or negative for the transgene are measured using fluorescent PCR products. Then, using probability and statistical distribution (Poisson distribution) analysis, the number of copies of the transgene present can be calculated.

What if there is no published copy number for my particular strain?

A previously published copy number for your strain is not required for our copy number testing service as long as controls of a known genotype (hemizygous or homozygous) are included with the first batch of samples. Mice that are hemizygous for the transgene should be readily identifiable as the transgene-positive offspring of a transgene-positive x Wildtype mating, however known homozygotes should also suffice. From these controls the haploid copy number of your mice can then be determined, and the genotypes of all other samples deduced. Please discuss with GRS to determine suitable controls.

However, if the copy number of your mice has been previously published, this will be a  valuable comparison for your results.

Can you test all types of Transgene modifications?

All varieties of transgenes should be suitable for copy number testing. During our test development phase we will research your strain and determine a suitable target sequence within the transgene to design the assay on.

Our copy number testing service may also be suitable for identifying off-target insertions from CRISPR modifications and for studies of copy number mutations of endogenous genes. Please contact GRS to discuss these applications.