IgG variable region and VH CDR3 diversity in unimmunized mice analyzed by massively parallel sequencing

Area of Science:

• Immunology
• Molecular Biology
• Genomics

Background:

• Hybridoma technology predominantly uses Balb/c mice for antigen-specific antibody generation.
• The germline variable gene repertoire in Balb/c mice is well-characterized.
• Limited information exists on the baseline IgG antibody repertoire in unimmunized mice.

Purpose of the Study:

• To assess the baseline expressed IgG antibody repertoire in young, unimmunized mice.
• To understand the natural variation and biases in antibody gene usage.
• To establish a baseline for comparison with antigen-specific antibody repertoires.

Main Methods:

• RNA isolation from splenic B-cells enriched for IgG expression in three mice.
• Amplification of heavy and light chain variable regions using distinct PCR primer sets.
• Deep sequencing (454 pyro-sequencing) and analysis of gene usage, redundancy, and VH CDR3 sequences.

Main Results:

• Highly skewed abundance in heavy and light chain variable gene usage was observed across all mice and primer sets.
• Significant overlap but also differences in gene profiles, indicating primer bias and inter-animal variation.
• VH CDR3 sequences were also highly skewed, reflecting individual antibody profiles with low redundancy across mice.

Conclusions:

• The background IgG antibody repertoire in young, unimmunized mice is highly skewed within individuals.
• The repertoire exhibits considerable diversity among different mice.
• This skewed and diverse pattern is similar to that observed in immunized mice.