Creating new and different strains for our clients and research projects is extremely exciting and rewarding. We at InVivo Biosystems have created over 3,000 confirmed C. elegans strains. These strains range from precise deletions (knockouts), to insertions (knock-ins), to single point mutations. It is imperative that these newly developed strains are made correctly.
So, how do we ensure that we deliver high quality C. elegans strains to our clients so that they are confident in the transgenic animals they receive? Every client project we take on goes through a rigorous, multi-step, screening verification process that confirms we are delivering the correct and accurate transgenic C. elegans strains to our clients each and every time!
Step 1: Quality Screening Design
The first step in most strain verification processes following micro-injections is an initial genotyping screen performed by polymerase chain reaction (PCR) and/or high resolution melt analysis (HRMA). However, before genotyping even begins, each project goes through an initial two-step screening design process. This step is extremely important as it allows for increased confidence in identifying the desired edit in our clients’ projects.
The screen design process begins by identifying primers that specifically target the locus of interest while also minimizing the possibility of non-specific binding. This step ensures that the primers are not binding elsewhere in the genome thus resulting in non-specific product amplification. Through the use of bioinformatics, we also verify that the designed primers will not bind to themselves or each other, or create additional secondary structures during the amplification process. If using HRMA as part of the screening process, predictive melt curve software is employed to ensure clear differentiation of mutant and wild-type alleles.
Once the screen design is complete, it is validated to be sure all possible errors are identified before the final screen design is confirmed. This emphasis on accuracy at the initiation of the design process promotes screening success and greatly reduces the possibility of rework or troubleshooting later in the transgenic build process.
Step 2: Genotyping and Sequence Confirmation of Potential Candidates
With the robust aforementioned screening method in place, potential target candidates can be identified with high confidence via PCR and HRMA. After potential candidates have been identified through genotyping, it is important to confirm that the desired edit in the animal integrated correctly. This is done within the second step in the process through DNA sequencing. As part of the screening design process, it is imperative that a sequencing PCR screen that amplifies the locus of interest was also meticulously designed and validated.
With this specific PCR product we can be confident that the proper edit and repair in the animal was made. The sequencing process further confirms the accuracy of the integration. If the sequencing of the target locus reveals any improper repair, point mutations, or any other unexpected abnormalities, the candidate is eliminated from consideration and additional transgenic candidates are pursued instead.
Step 3: Isolate, Genotype, and Confirm Progeny
The third step of the confirmation process is to isolate progeny from sequence confirmed candidates. This step is critical to ensure that the animal and its progeny are indeed homozygous mutants at the locus of interest. The candidate’s progeny are also run through the initial screening process to confirm their genotyping results are in agreement with the parental screen. This imparts further confidence and allows us to once again verify that the animal is the correct candidate and ready to be moved to the authentication step.
Step 4: Authentication of Candidate C. elegans Strain
Once the final screening process is complete, the candidate transgenic strain goes through a final authentication step. All screening and sequencing data is compiled and reviewed by the laboratory supervisor for final approval. At this point in the process, the animal is given a unique strain identification number and prepared for delivery to the client.
While many of the steps in our screening process may sound or seem redundant, we feel that this level of care, accuracy, and attention to detail is of great benefit to our clients. We believe that when our clients receive their transgenic animals from InVivo Biosystems, they can be confident that their project has gone through an extremely rigorous screening and validation process. This allows our clients to perform their experiments with confidence and focus on answering all of their research questions. It also gives our clients the peace of mind that if they ever need additional transgenics for future projects, they will come back to InVivo Biosystems with the expectation to receive the same great products and services.