Single cell measurement of telomerase expression and splicing using microfluidic emulsion cultures.

Telomerase is a reverse transcriptase that maintains telomeres on the ends of chromosomes, allowing rapidly dividing cells to proliferate while avoiding senescence and apoptosis. Understanding telomerase gene expression and splicing at the single cell level could yield insights into the roles of telomerase during normal cell growth as well as cancer development. Here we use droplet-based single cell culture followed by single cell or colony transcript abundance analysis to investigate the relationship between cell growth and transcript abundance of the telomerase genes encoding the RNA component (hTR) and protein component (hTERT) as well as hTERT splicing. Jurkat and K562 cells were examined under normal cell culture conditions and during exposure to curcumin, a natural compound with anti-carcinogenic and telomerase activity-reducing properties. Individual cells predominantly express single hTERT splice variants, with the α+/β- variant exhibiting significant transcript abundance bimodality that is sustained through cell division. Sub-lethal curcumin exposure results in reduced bimodality of all hTERT splice variants and significant upregulation of alpha splicing, suggesting a possible role in cellular stress response. The single cell culture and transcript abundance analysis method presented here provides the tools necessary for multiparameter single cell analysis which will be critical for understanding phenotypes of heterogeneous cell populations, disease cell populations and their drug response

Exhibit 6 Part 3 from R. Hart

Exhibit 6, part 3, A History of Coeur d\u27Alene Tribal Water Use 1780-1915, attached to the sworn affidavit of R. Hart

Exhibit 6 Part 2 from R. Hart

Exhibit 6, part 2, A History of Coeur d\u27Alene Tribal Water Use 1780-1915, attached to the sworn affidavit of R. Hart

Affidavit from R. Hart

The sworn affidavit of Richard Hart

Exhibit 6 Part 1 from R. Hart

Exhibit 6, part 1, A History of Coeur d\u27Alene Tribal Water Use 1780-1915, attached to the sworn affidavit of R. Hart

Exhibit 6 Part 4 from R. Hart

Exhibit 6, part 4, A History of Coeur d\u27Alene Tribal Water Use 1780-1915, attached to the sworn affidavit of R. Hart

Exhibit 6 Part 3 from R. Hart

Exhibit 6, part 3, A History of Coeur d\u27Alene Tribal Water Use 1780-1915, attached to the sworn affidavit of R. Hart

Exhibit 6 Part 1 from R. Hart

Exhibit 6, part 1, A History of Coeur d\u27Alene Tribal Water Use 1780-1915, attached to the sworn affidavit of R. Hart