Event Title

Quantification of Xpo7 alternative splice variant using quantitative polymerase chain reaction

Location

Edwards 107

Document Type

Oral/PowerPoint Presentation (10 minutes and 5 minute Q and A)

Description

Erythroid terminal differentiation (ETD) describes the latest stages of erythrocyte differentiation where precursor cells undergo biochemical and structural changes to develop into functional red blood cells. Previous studies identified specific up-regulation of a gene called exportin 7 (Xpo7), a protein involved in nuclear export, during ETD. It is hypothesized that Xpo7 has a critical role in shuttling nuclear proteins out of the nucleus and causing chromatin condensation that ultimately leads to nuclear extrusion of differentiating cells, a critical step of erythropoiesis.

Xpo7 was amplified using polymerase chain reaction (PCR) to obtain complementary DNA for the coding region of Xpo7 messenger RNA (mRNA). It was then cloned and ligated into an inducible expression vector. Upon sequencing of the cloned fragment to confirm presence of Xpo7, BLAST showed the absence of exon 11 in Xpo7 sequence, suggesting an alternative splicing event. The size of this exon is 172 base pairs and the splicing of this exon causes a frame-shift in the open reading frame producing Xpo7 protein with truncated C terminus. Translation of the alternative mRNA sequence with spliced exon 11 reveals an altered protein sequence with 26 different amino acids beyond the spliced site as compared to the normal mRNA. The presence of an alternative variant of Xpo7 with exon 11 spliced in mature mRNA in mice has not been previously reported, and thus it may play a unique role in ETD. Currently, the determination of the relative amount of the Xpo7 alternative splice variant is being done using quantitative PCR.

Start Date

April 2014

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Apr 26th, 2:30 PM

Quantification of Xpo7 alternative splice variant using quantitative polymerase chain reaction

Edwards 107

Erythroid terminal differentiation (ETD) describes the latest stages of erythrocyte differentiation where precursor cells undergo biochemical and structural changes to develop into functional red blood cells. Previous studies identified specific up-regulation of a gene called exportin 7 (Xpo7), a protein involved in nuclear export, during ETD. It is hypothesized that Xpo7 has a critical role in shuttling nuclear proteins out of the nucleus and causing chromatin condensation that ultimately leads to nuclear extrusion of differentiating cells, a critical step of erythropoiesis.

Xpo7 was amplified using polymerase chain reaction (PCR) to obtain complementary DNA for the coding region of Xpo7 messenger RNA (mRNA). It was then cloned and ligated into an inducible expression vector. Upon sequencing of the cloned fragment to confirm presence of Xpo7, BLAST showed the absence of exon 11 in Xpo7 sequence, suggesting an alternative splicing event. The size of this exon is 172 base pairs and the splicing of this exon causes a frame-shift in the open reading frame producing Xpo7 protein with truncated C terminus. Translation of the alternative mRNA sequence with spliced exon 11 reveals an altered protein sequence with 26 different amino acids beyond the spliced site as compared to the normal mRNA. The presence of an alternative variant of Xpo7 with exon 11 spliced in mature mRNA in mice has not been previously reported, and thus it may play a unique role in ETD. Currently, the determination of the relative amount of the Xpo7 alternative splice variant is being done using quantitative PCR.