How does the cell line genotype name associate with the DNA sequence?
What is the exact DNA change in the cell line?
Where is the mutation in my gene/protein?
What makes up a Sequence Map file?
What are the contents of the Sequence Map files? Where can I find more detailed information about the mutation and how do I read the Map files?
How did you select relevant mutations?
The mutation I am looking for is not available. Are you planning to add additional cell lines?
Where can I find the CRISPR sequences and associated information used for creating the mutants?
Where can I find the DNA primers that are used for sequence validation after gene editing?
Why is using the revertant a better control compared to using the parental cell line?
From which of the cell lines (heterozygous or homozygous mutation-carrying) has the revertant been created?
What is the difference between clones A and B in your catalog?

1. How does the cell line genotype name associate with the DNA sequence?

The DNA sequence in the parental cell line is considered the reference and the wild type (WT) allele designation reflects this. The gene-edited variant allele is noted as SNV (single nucleotide variant). The revertant allele (Rev or REV) is the same sequence as the parental allele (WT) and was derived from the gene editing process of modifying the SNV allele back to wild type.

2. What is the exact DNA change in the cell line?

Each cell line product page contains downloadable Sequence Map files. The exact DNA change is annotated in the Features sections of these design files. The information is readily found in the SNV subsection of the Features section which specifies the nucleotide base and its relationship to WT, REV or SNV cell line name nomenclature.

3. Where is the mutation in my gene/protein?

Most mutations in this catalog are associated with single nucleotide variants defined by the NIH National Center for Biotechnology dbSNP' database. This unique identifier is present in the Genomic Features details of each cell line's details page. The exact genomic coordinate of the variant is indicated in the Genomic Features section as well. Finally, the Sequence Map files for each cell line contain rich data and visual presentations of the gene features, gene editing tools, and genotyping primers, as well as DNA/Protein information.

4. What makes up a Sequence Map file?

The Sequence Map for each cell line is created using SnapGene software by importing the current human reference genomic sequence gene (GRCh38), RNA and protein annotations. The cell line-specific gene editing reagents, genotyping primer sequences, Sanger Sequencing primer(s), and variant specifics are included and presented in a comprehensive visual map. These Map files are available in three different file types: SnapGene, PDF and GenBank. NOTE: The reference sequence in these Maps does NOT represent the sequence of the KOLF2.1J parental genome sequence.

5. What are the contents of the Sequence Map files? Where can I find more detailed information about the mutation and how do I read the Map files?

You can find more detailed information in the Sequence Map document. The SnapGene® software package has been used to create the Sequence Map files (snapgene.com). A free read-only version of the software can be downloaded for viewing the SnapGene file type. In the PDF output of the SnapGene file type map, the first page displays a general overview of the respective gene derived from the human reference genome database (gray). The canonical primary Ensembl RNA transcript used in this Map is shown in orange, the protein coding sequence is displayed in purple, the donor template is shown in sky blue, the protospacer in red, and the SNV in orange. The subsequent pages show the detailed sequence in base pair resolution with the respective features annotated including the sequence of the PCR primer pair used for Sanger sequencing validation. The base in the reference sequence targeted for change is colored in red letters.

6. How did you select relevant mutations?

Genes and variants were selected by the researchers composing the NIH iNDI. The Ramos et al, 2021 publication in Neuron provides a more detailed context.

7. The mutation I am looking for is not available. Are you planning to add additional cell lines?

We will be adding additional cell lines to our existing Distribution Sets once available, including whole gene knockouts and SNV-containing and wild type genes fused to the multifunctional HaloTag® sequence. The aim is to generate cell lines for 134 variants across 73 ADRD genes. Please email micetech@jax.org if you are looking for a variant that is currently not covered.

8. Where can I find the CRISPR sequences and associated information used for creating the mutants?

You can find this information on our website catalog by clicking on 'view more' under 'gene editing sequences' of the respective cell line. The protospacer sequence of the gRNAs is also included in the Sequence Map files.

9. Where can I find the DNA primers that are used for sequence validation after gene editing?

You can find this information on our website catalog by clicking on 'view more' under 'DNA Primers for Sequence Validation' of the respective cell line. The primers used for PCR and Sanger sequence validation are also included in the Sequence Map files.

10. Why is using the revertant a better control compared to using the parental cell line?

The use of revertant lines may allow discrimination of phenotypes caused by the mutation to not be confused by off-target consequences of the gene editing process to create SNVs. Undetected off-target mutations are unlikely to be correctly gene engineered back to the KOLF2.1J “reference” state (Wildtype=WT). If the phenotypic signal is not suppressed in the revertant, it is possible the experimental signal detected is not caused by the expected SNV but by a second mutation.

11. From which of the cell lines (heterozygous or homozygous mutation-carrying) has the revertant been created?

In the 'Genetics' section of each cell line's details page, the genotype of the revertant's parent is noted in the 'Origin of Revertant'. For much of the catalog, the autosomal dominant mutations have reverted heterozygous lines, while for recessive mutations, we have engineered homozygous lines back to the wildtype (KOLF2.1J) sequence. Some JAX Distribution groups will have revertants created from both heterozygous (SNV/WT) and homozygous (SNV/SNV) cell lines.

12. What is the difference between clones A and B in your catalog?

In the few cases where both clones A and B are listed, please note that clone B is an independent clone, generated using different gene-editing reagents than clone A. Using more than one clone can support efforts to assess and improve reproducibility in experimental results.

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