Cytogenetic, Genomic, and Functional Characterization of Pituitary Gonadotrope Cell Lines.

LT2 and T3-1 are important, widely studied cell line models for the pituitary gonadotropes that were generated by targeted tumorigenesis in transgenic mice. LT2 cells are more mature gonadotrope precursors than T3-1 cells. Microsatellite authentication patterns, chromosomal characteristics, and their intercellular variation have not been reported. We performed microsatellite and cytogenetic analysis of both cell types at early passage numbers. Short tandem repeat (STR) profiling was consistent with a mixed C57BL/6J × BALB/cJ genetic background, with distinct patterns for each cell type. Spectral karyotyping in T3-1 cells revealed cell-to-cell variation in chromosome composition and pseudodiploidy. In LT2 cells, chromosome counting and karyotyping demonstrated pseudotriploidy and high chromosomal variation among cells. Chromosome copy number variation was confirmed by single-cell DNA sequencing. Chromosomal compositions were consistent with a male sex for T3-1 and a female sex for LT2 cells. Among LT2 stocks used in multiple laboratories, we detected two genetically similar but distinguishable lines via STR authentication, LT2a and LT2b. The two lines differed in morphological appearance, with LT2a having significantly smaller cell and nucleus areas. Analysis of immediate early gene and gonadotropin subunit gene expression revealed variations in basal expression and responses to continuous and pulsatile GnRH stimulation. LT2a showed higher basal levels of , , and but lower induction. induction reached significance only in LT2b cells. Our study highlights the heterogeneity in gonadotrope cell line genomes and provides reference STR authentication patterns that can be monitored to improve experimental reproducibility and facilitate comparisons of results within and across laboratories.