Browsing by Subject "CRISPR/CAS"

Lethal congenital contracture syndrome 1 (LCCS1) is a severe developmental disorder that is part of the Finnish disease heritage. The affected foetuses die in utero and show a lack of motor neurons accompanied by severe atrophy of the ventral spinal cord and muscles and severe contracture of joints, which result in a lack of involuntary movements. Other associated symptoms include hydrops, micrognatia (small jaw), pulmonary hypodysplasia and small size. The syndrome leads to prenatal death before 32nd gestational week, but the cause remains elusive. LCCS1 is caused by a homozygous mutation, FINmajor, in GLE1 RNA export mediator (GLE1) -gene. The mutation is a c.432- 10A > G substitution at the border of intron three and exon four, resulting in a new splice acceptor site 10 nucleotides upstream of the intron-exon junction. This results in aberrant splicing and nine extra nucleotides in the mRNA, corresponding to three extra amino acids in the GLE1 protein of the affected individuals. GLE1 is an important player in RNA biology in cells. In humans it has two isoforms, GLE1A and GLE1B, that have distinct roles. While GLE1A plays a role in cells’ stress response by mediating the formation and disassembly of stress granules in the cytoplasm, GLE1B is found at the nuclear envelope where it mediates mRNA transport from the nucleus to the cytoplasm. Earlier studies using HeLa cells and zebrafish have demonstrated that FINmajor in GLE1 knock-down background disrupts the mRNA export from nucleus to cytoplasm and leads to apoptosis in neural precursors and abnormal arborization of motor neurons, thus mimicking some of the phenotypic features observed in human patients. Kuure group has generated a mouse model with endogenous FINmajor, but it fails to morphologically phenocopy the human disorder (unpublished data). Importantly, human-origin non-cancerous models with endogenous FINmajor mutation have not yet been used in the studies of GLE1 and LCCS1. My aim in the thesis was to create a human embryonic stem (hES) cell line carrying homozygous FINmajor mutation in its genome. A relatively new tool, CRISPR/CAS -system allows for precise genetic engineering in a variety of model organisms. By optimizing the system to efficiently edit the GLE1 gene, I was able to introduce FINmajor mutation in hES cells, creating a new model system to study this disorder. While previous models had relied on temporary silencing of the gene with morpholinos or siRNAs, the hES cell line with GLE1 FINmajor mutation will for the first time give insights on how the mutation affects cellular functions, mRNA biology and cell differentiation in LCCS1. This way the hES cell line I have generated will yield new information on the development, progression and manifestation of the syndrome to better understand its mechanisms.