Peter Bauer

Immunocytochemical characterization of in vitro PKH26-labelled and intracerebrally transplanted neonatal cells

The lipophilic dye PKH26 that binds irreversibly to cell membranes has been used to label various cell types in vitro prior to transplantation in order to recognize grafted cells posttransplantationally in the host tissue by fluorescence microscopy. The purpose of the present study was to optimize immunocytochemical staining procedures for PKH26-containing specimens in cell culture and after transplantation into rat brain. We demonstrated that freeze-thawing allowed for proper immunostaining of intracellular epitopes whereas PKH26-labelling was preserved. PKH26-labelled donor cells were detectable at least up to 4 months after transplantation in the host brain.

Expanding the clinical and genetic spectra of NKX6-2-related disorder

Hypomyelinating leukodystrophies (HLDs) affect the white matter of the central nervous system and manifest as neurological disorders. They are genetically heterogeneous. Very recently, biallelic variants in NKX6‐2 have been suggested to cause a novel form of autosomal recessive HLD. Using whole‐exome or whole‐genome sequencing, we identified the previously reported c.196delC and c.487C>G variants in NKX6‐2 in 3 and 2 unrelated index cases, respectively; the novel c.608G>A variant was identified in a sixth patient. All variants were homozygous in affected family members only. Our patients share a primary diagnosis of psychomotor delay, and they show spastic quadriparesis, nystagmus and hypotonia. Seizures and dysmorphic features (observed in 2 families each) represent an addition to the phenotype, while developmental regression (observed in 3 families) appears to be a notable and previously underestimated clinical feature. Our findings extend the clinical and mutational spectra associated with this novel form of HLD. Comparative analysis of our 10 patients and the 15 reported previously did, however, not reveal clear evidence for a genotype‐phenotype correlation.

Amplification of Nucleic Acids by Polymerase Chain Reaction: Overview on Principles and Applications

The polymerase chain reaction (PCR) is a powerfulin vitromethod in molecular biology for selective, highly specific and exceptionally efficient amplification of nucleic acid sequences. In the 10 years since the first publication on PCR (Saiki et al., 1985) this method has grown to rival in popularity traditional microbiological, genetic and technical procedures for cloning, sequencing, gene detection and related procedures. Furthermore, in the meantime PCR and all of its different applications are rapid and convenient alternatives to traditional procedures such as blotting technologies, conventional hybridization and molecular cloning. Initially, PCR was a rather complex and tricky generic procedure applied to basic research problems in molecular biology. It has developed into a simple, multipurpose procedure more or less optimized for diverse applications in nearly every biological discipline and commercial area. There are frequent instances of PCR techniques having passed into the service laboratory environment. These service laboratories are providing a broad range of diagnostic tests mainly covering medical and forensic applications, but also environmental, agricultural and veterinary topics.