|Lysyl oxidases: Cloning and characterization of the fourth and the fifth human lysyl oxidase isoenzymes, and the consequences of a targeted inactivation of the first described lysyl oxidase isoenzyme in mice|
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A genomic clone containing murine Lox gene sequences was isolated from the 129SV library using human LOX cDNA (Hämäläinen et al. 1991) as a probe. A 10.2 kb fragment containing the 5’end of the gene was subcloned into BlueScript plasmid (Promega), and the loxP sequence was inserted into a NsiI site located about 600 bp upstream of the translation start codon in front of the predicted transcription start sites (Contente et al. 1993). A HSV-tk-neor selection cassette flanked by loxP sites was inserted into a BamHI site located 90 bp downstream of the first exon (see Figure 1A in III).
The linearized targeting construct was electroporated into R1 embryonic stem (ES) cells (Nagy et al. 1993). Genomic DNA from the G418-resistant ES clones was digested with SpeI and hybridized with a B1/SpeI external probe. Cells from correctly targeted ES clones were electroporated with a pIC-Cre plasmid and grown in the presence of Gancyclovir. DNA from clones that had survived selection was digested with NsiI and hybridized with the B1-E1/E1 probe. Cells in which the selection cassette and the first exon of the Lox gene had been deleted were injected into C57BL/6 blastocysts, which were then implanted into pseudopregnant females. The resulting chimeras were bred with C57BL/6 mice to produce heterozygous mutant mice. By using heterozygous siblings, mutant embryos were obtained. Embryos were genotyped by Southern blotting, and total RNA was extracted from them. Northern blots from extracted total RNA were prepared by routine methods (see Figure 1 in III).
Tissue samples were fixed overnight in 10% buffered formalin and embedded in paraffin. Sections were stained with hematoxylin-eosin and Masson’s trichrome. The hematoxylin-eosin-stained aorta sections were examined under ultraviolet light. Processing and sectioning of Lox+/+ and Lox-/- samples were performed parallelly and were inspected without knowledge of the genotype. The diameter of the aortic lumen and wall thickness were measured from the proximal part of the descending aorta. Student’s t-test were used for establishing significant differences among groups.
Biopsies from the descending aorta of E18.5 embryos were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, postfixed in 1% osmium tetroxide, dehydrated in acetone, and embedded in Epon LX112. Thin sections were cut with a Reichert Ultracut ultramicrotome and examined in a Phillips CM100 transmission electron microscope.
Anesthesized embryos were examined using an Acuson Sequoia 512 Doppler ultrasonograph with a 13 MHz linear probe. The embryonic heart was identified by color Doppler, and the sample volume of the pulsed Doppler was placed over it. The length of the sample volume was adjusted to cover the entire heart, and the high pass filter was set at its minimum. Different views of the heart were examined to minimize the angle between the Doppler beam and the inflow and outflow regions and to obtain their maximal velocities. The maximal inflow and outflow velocities were recorded using a sweep speed of 100 mm/s. Different vessels were located on the sagittal view of the embryo with the help of color Doppler, and blood velocity waveforms were obtained by the pulsed Doppler method (see Figure 4A in III). Immediately after the ultrasonographic examination, the dam was sacrificed, the abdomen carefully opened, and the embryos identified according to their location in ultrasonography.
The ultrasonographic data were analysed using the cardiovascular measurement package included with the ultrasound equipment. Time-velocity-integrals were measured from the inflow and outflow blood velocity waveforms by planimetry of the area underneath the Doppler spectrum. The fetal heart rate was obtained, and the inflow and outflow mean velocities were calculated. Pulsatility index values were obtained from the descending aorta, intracranial arteries, and umbilical artery blood velocity waveforms, and pulsatility index values for veins were calculated from the ductus venosus blood velocity waveforms. The Doppler ultrasonographic parameters were analyzed statistically using Student’s t-test.