Abstract
Amyotrophic Lateral Sclerosis (ALS) is a devastating adult onset neurodegenerative disease affecting both upper and lower motor neurons. TDP-43, encoded by the TARDBP gene, was identified as a component of motor neuron cytoplasmic inclusions in both familial and sporadic ALS and has become a pathological signature of the disease. TDP-43 is a nuclear protein involved in RNA metabolism, however in ALS, TDP-43 is mislocalized to the cytoplasm of affected motor neurons, suggesting that disease might be caused by TDP-43 loss of function. To investigate this hypothesis, we attempted to generate a mouse conditional knockout of the Tardbp gene using the classical Cre-loxP technology. Even though heterozygote mice for the targeted allele were successfully generated, we were unable to obtain homozygotes. Here we show that although the targeting vector was specifically designed to not overlap with Tardbp adjacent genes, the homologous recombination event affected the expression of a downstream gene, Masp2. This may explain the inability to obtain homozygote mice with targeted Tardbp.
Highlights
Amyotrophic Lateral Sclerosis (ALS) is an adult-onset and invariably fatal neurodegenerative disease characterized by the loss of motor neurons in the brain and spinal cord
A neomycin resistance cassette flanked by FLP recognition target (FRT) sites was amplified by Polymerase Chain Reaction (PCR) and integrated downstream of Tardbp exon 3
In order to remove the flipped neomycin selection cassette, F1 Tardbp targeted mice were crossed to Actin-FLP mice and neomycin excision was verified by PCR amplification using primers flanking the FRT sites (Figure 1C)
Summary
Amyotrophic Lateral Sclerosis (ALS) is an adult-onset and invariably fatal neurodegenerative disease characterized by the loss of motor neurons in the brain and spinal cord. The cytoplasmic location of pathological TDP-43 suggests that disease might be caused by a loss of nuclear function To this end, Tardbp knockout mice have been generated to investigate this loss-of-function hypothesis, homozygote mice were embryonic lethal, dying between 3.5 to 8.5 days of embryonic development, while heterozygotes did not display any motor neuron pathology [11,12]. We successfully generated viable and fertile heterozygote mice with the targeted allele, we were unable to obtain homozygotes This was not due to effects of the targeting event on reducing TDP-43 expression, thereby mimicking a knockout mouse, but instead we show that the targeting event affected the expression of a downstream gene, Masp. This effect on Masp could underlie the inability to obtain homozygous mice with targeted Tardbp
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