Gene targeting--homologous recombination of DNA sequences residing in the chromosome with newly introduced DNA sequences--in mouse embryo-derived stem cells promises to provide a means to generate mice of any desired genotype. We describe a positive nd negative selection procedure that enriches 2,000-fold for those cells that contain a targeted mutation. The procedure was applied to the isolation of hprt- and int-2- mutants, but it should be applicable to any gene.
The int-1 proto-oncogene was first identified as a gene activated in virally induced mouse mammary tumours. Expression studies, however, suggest that the normal function of this gene may be in spermatogenesis and in the development of the central nervous system. Genes sharing sequence similarity with int-1 have been found throughout the animal kingdom. For example, int-1 has 54% amino-acid identity to the Drosophila segment polarity gene wingless (wg). Both the int-1 and wg gene products seem to be secreted proteins, presumably involved in cell-cell signalling. We have now explored the function of int-1 in the mouse by disrupting one of the two int-1 alleles in mouse embryo-derived stem cells using positive-negative selection. This cell line was used to generate a chimaeric mouse that transmitted the mutant allele to its progeny. Mice heterozygous for the int-1 null mutation are normal and fertile, whereas mice homozygous for the mutation may exhibit a range of phenotypes from death before birth to survival with severe ataxia. The latter pathology in mice and humans is often associated with defects in the cerebellum. Examination of int-1-/int-1- mice at several stages of embryogenesis revealed severe abnormalities in the development of the mesencephalon and metencephalon indicating a prominent role for the int-1 protein is in the induction of the mesencephalon and cerebellum.
METHODS Migration AssaysMigration assays were performed as described (3)(4)(5). Briefly, 16 h before the assay, 80% confluent 75 cm 2 flasks (Corning Costar) of human microvessel endothelial cells (HMVEC; Cambrex, Walkersville, MD), human coronary artery endothelial cells (HCAEC; Cambrex), human umbilical artery endothelial cells (HUAEC; Promocell, Heidelburg, Germany), or human umbilical vein endothelial cells (HUVEC; Promocell), were washed with Hank's Balanced Salt Solution (HBSS, Invitrogen) and serum-starved overnight in endothelial basal media (EBM-2, Cambrex) with 0.1% fatty-acid-free BSA (Sigma) and 0.5% fetal calf serum (FCS, Hyclone). The following day cells were lifted with Trypsin/EDTA solution (Promocell), mixed with an equal volume Trypsin Neutralization Solution (Promocell), and washed 3 times in migration media (EBM-2 with 0.1% fatty-acid-free BSA and 0.2% FCS). Cells were resuspended at a density of 1.5×10 6 cells/ml and were allowed to recover for 1 h at 37°C (5% CO 2 ). 3.75 × 10 4 cells were plated into each well of a 48-well Boyden chamber apparatus (NeuroProbe, Cabin John, MD), and the wells were overlayed with an 8 μm pore polycarbonate membrane (NeuroProbe) that had been previously coated with 50 μg/ml human fibronectin (Biomedical Technologies, Inc., Stoughton, MA). Experiments performed with membranes coated with acetylated 1% gelatin from porcine skin (Sigma, St. Louis, MO) gave similar results. The apparatus was assembled and stored inverted at 37°C (5% CO 2 ) for 2 h. The apparatus was then re-inverted and 52 μl of purified chemoattractants [murine netrin-1 (R&D Systems, Minneapolis, MN), chicken netrin-2 (R&D Systems), murine netrin-4 (R&D Systems), murine netrin-G1a (R&D Systems), human VEGF 165 (R&D Systems), or control/ migration media (EBM-2 with 0.1% fatty-acid-free BSA and 0.2% FCS) were added to the upper chambers, and the migration was allowed to proceed for 2 h at 37°C (5% CO 2 ). The membranes were then removed, fixed in methanol, stained with a Hema 3 stain set (Fisher Scientific, Pittsburgh, PA), and placed (migrated-side down) onto 50 × 75 mm glass slides. Before 90% mounting medium (in xylenes) and coverslips were applied, the non-migrated cells were removed from the exposed (non-migrated) side of the membrane with a moistened swab. Cells present on the migrated side of the membrane were manually counted (three random 200× fields per well), and data points for each experiment represent the average number of migrated cells from six separate wells (three 200× fields counted per well).Another method was employed in a separate laboratory to evaluate the effects of the netrins on mouse (MS1) endothelial cells (ATCC, Manassas, VA) using a modified Boyden chamber assay as described previously (6). Briefly, a 5 μm-polycarbonate filter (Poretics) was placed between upper and lower chamber. Cell suspensions (5×10 4 cells/well) were placed in the upper chamber, and the lower chamber was filled with serum-free medium containing
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