Abstract
Recently, it has been found that spontaneous mutation Lx (polydactyly-luxate syndrome) in the rat is determined by deletion of a conserved intronic sequence of the Plzf (Promyelocytic leukemia zinc finger protein) gene. In addition, Plzf is a prominent candidate gene for quantitative trait loci (QTLs) associated with cardiac hypertrophy and fibrosis in the spontaneously hypertensive rat (SHR). In the current study, we tested the effects of Plzf gene targeting in the SHR using TALENs (transcription activator-like effector nucleases). SHR ova were microinjected with constructs pTAL438/439 coding for a sequence-specific endonuclease that binds to target sequence in the first coding exon of the Plzf gene. Out of 43 animals born after microinjection, we detected a single male founder. Sequence analysis revealed a deletion of G that resulted in frame shift mutation starting in codon 31 and causing a premature stop codon at position of amino acid 58. The Plzftm1Ipcv allele is semi-lethal since approximately 95% of newborn homozygous animals died perinatally. All homozygous animals exhibited manifestations of a caudal regression syndrome including tail anomalies and serious size reduction and deformities of long bones, and oligo- or polydactyly on the hindlimbs. The heterozygous animals only exhibited the tail anomalies. Impaired development of the urinary tract was also revealed: one homozygous and one heterozygous rat exhibited a vesico-ureteric reflux with enormous dilatation of ureters and renal pelvis. In the homozygote, this was combined with a hypoplastic kidney. These results provide evidence for the important role of Plzf gene during development of the caudal part of a body—column vertebrae, hindlimbs and urinary system in the rat.
Highlights
Deletions of the chromosomal region 11q23 are known in human resulting in a phenotype including mental retardation, craniofacial dysmorphism, microcephaly and short stature
We bred the founder with spontaneously hypertensive rat (SHR) to generate more heterozygous animals, and intercrossed the heterozygotes to obtain knock-out homozygotes
No promyelocytic leukaemia zinc finger (PLZF) protein was detected in the tissues isolated from perinatal SHR-Plzf-/- homozygotes (Fig 1F)
Summary
Deletions of the chromosomal region 11q23 are known in human resulting in a phenotype including mental retardation, craniofacial dysmorphism, microcephaly and short stature. Within the chromosomal region 11q23, the promyelocytic leukaemia zinc finger (PLZF) gene encodes a DNA sequence-specific transcriptional repressor (OMIM 176797). A similar mutation exists in animal models. In Wistar outbred rats, spontaneous mutation Lx (polydactyly-luxate syndrome) was originally detected and fixed in the PD/Cub (polydactylous) inbred strain and transferred to genetic backgrounds of the BN-Lx (Brown Norway) and SHR-Lx (spontaneously hypertensive rat) congenic strains [1,2]. Based on the identification of the mouse lu (Green's luxoid) spontaneous mutation as a nonsense point mutation in the Plzf (Promyelocytic leukemia zinc finger protein) gene [3,4] and conservation of orthologous regions of mouse chromosome 9 and rat chromosome 8, that include lu and Lx genes, respectively, Plzf was an obvious candidate for the Lx mutation. PLZF is a multifunctional transcriptional repressor involved in major biological processes during development, for instance in stem cell self-renewal, including hematopoietic stem cells, neural progenitor cells or spermatogonial progenitor cells and in stem cell differentiation, including myeloid differentiation, limb bud development, osteogenesis, chondrogenesis or lymphoid differentiation [6]
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