Transgene Mapping (Locus Amplification & Sequencing)

For transgenic cell lines and transgenic animals generated via many methods, the integration site is random and, in most cases, not known. Integration of a transgene can disrupt an endogenous gene, potentially interfering with interpretation of the phenotype. In addition, knowledge of where the transgene is integrated is important for planning of crosses between animals carrying a conditional allele and a given Cre allele in case the alleles are on the same chromosome. When the transgene insertion site is unknown, zygosity is determined by expensive quantitative PCR-based approach. These limitations often force investigators to maintain transgenic models or cell lines in a hemizygous state, which may lead to less than desired expression levels of the transgene and make it less efficient and more costly. Creative Bioarray has developed the Transgene Mapping (Locus Amplification & Sequencing) analysis, which is a powerful tool to sequence transgenes and their integration sites.

Transgene Mapping (Locus Amplification & Sequencing) analysis combines the next generation sequencing to selectively amplify and sequence the transgene and surrounding genomic region. This method not only uncovers insertion sites and sequences of integrated transgenes, but also enables detection of single nucleotide variations, structural variations, and transgene-transgene fusions.

Transgene Mapping (Locus Amplification & Sequencing) in the Cell Line Development Process

Transgene locus amplification & Sequencing presents a cost-effective and high-quality alternative to conventional approaches to control the quality of transgenic cells.

Transgene Mapping (Locus Amplification & Sequencing) analysis can:

  • Identify the transgene integration site(s)
  • Detect structural changes in the host DNA at the transgene integration site(s)
  • Sequence the entire transgene and detect any sequence variants as well as structural changes within the transgene
  • Provide an estimation of the transgene copy number

Overview of different stages of cell line development (in circles) and the purpose of Transgene Mapping (Locus Amplification and Sequencing) analyses (A-E).Figure 1: Overview of different stages of cell line development (in circles) and the purpose of Transgene Mapping (Locus Amplification & Sequencing) analyses (A-E).

Transgene Mapping (Locus Amplification & Sequencing) in the Transgenic Animal Process:

Transgene Mapping (Locus Amplification & Sequencing) analysis can:

  • Better correlation of phenotypes with transgene expression
  • Ability to determine zygosity by genotyping assay and more cost-effective management of transgenic strains
  • Enhanced predictability of transgene segregation when breeding and intercrossing
  • Awareness of any potential disruption of the regulatory or coding region of a critical endogenous gene

Transgene integration is random.Figure 2. Transgene integration is random.

Features of Creative Bioarray's Transgene Mapping (Locus Amplification & Sequencing) Service:

  • Identify the integration site(s)
  • Identify the integrity of the transgene (sequence variants and structural variants)
  • Identify the integrity of the host genome in the vicinity of the integration sites
  • Identify the breakpoint sequences between the genome and the vector
  • Identify vector-vector fusion reads to indicate the presence of concatemers
  • Estimate the transgene copy number
  • Allow the comprehensive genetic characterization of CHO cell lines

Advantages of Transgene Mapping (Locus Amplification & Sequencing) to conventional methods of transgene mapping

Advantages of Transgene Mapping (Locus Amplification and Sequencing) to conventional methods of transgene mapping

Deliverable

  • Full scope support service-from sample preparation through to the final report
  • Detailed report of insertion and transgene position
  • Expert interpretation specific to a new transgenic line

Quotation and Ordering

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