Bright fluorescent probes for chromosome enumeration and controls for locus-specific detection.
GeneCopoeia's VividFISH™ fluorescence in situ hybridization (FISH) chromosome probes are the ideal tools for chromosome detection. FISH chromosome probes are extremely valuable for many applications, including:
- Exceptionally bright fluorescent probes make chromosome detection easy and convenient.
- Each probe available for fluorescence in multiple colors.
- Great for use in chromosome copy number determination for genome editing applications.
Immortalized mammalian cell lines, while providing convenient model systems for biomedical and pharmaceutical research, are often aneuploid. For example, the commonly-used human embryonic kidney cell line HEK293 is hypotriploid, with a modal chromosomal number of 64. Further, the ploidy of HEK293 and some other cell lines is not uniform among cells in a population. This deviation of ploidy from the normal diploid state in immortalized cell lines poses a challenge for genome editing applications, such as gene knockout and mutagenesis, which often require complete elimination of wild type gene function. Therefore, knowledge of the number of copies of a targeted gene to be edited could signicantly benefit a cell line-based genome editing project.
GeneCopoeia's VividFISH™ chromosome enumeration probes are powerful tools for determining target gene copy number in genome editing applications. Using VividFISH™ probes to determine target chromosome number in a cell line before undergoing a genome editing project can help guide clone screening for desired modifications. To illustrate this concept, GeneCopoeia used a VividFISH™ chromosome enumeration probe to determine chromosome copy number in the human synovial fibroblast cell line MH7A, in which the X chromosome HDAC6 gene was being knocked out using CRISPR. Using VivdFISH™, we determined that MH7A cells carry five copies of the X chromosome (Figure 1A). This information helped us identify single clones that contained all five copies of the HDAC6 gene carrying CRISPR-mediated frameshift mutations (Figure1B).
Figure 1. Using VividFISH™ chromosome enumeration probes to identify pentalleleic gene knockout in a mammalian cell line. A. Fluorescence in situ hybridization (FISH) analysis reveals the presence of 5 copies of the HDAC6 gene in MH7A cells. Unmodified MH7A cells (top) and the diploid human cell line HT1080 (bottom) were hybridized with VividFISH™ probes recognizing the X chromosome (green), and the Y chromosome (orange). Only the male cell line HT1080 hybridizes with the Y chromosome probe. Nuclei were stained with DAPI (blue). B. Sequence analysis from two single clones following transfection of MH7A cells with GeneCopoeia Genome-CRISPTM sgRNA-expressing plasmids targeting HDAC6 (NCBI geneID:10013). Each clone carries 5 distinct frame shift alleles generated in a single transfection.