A 'rescuable' plasmid containing globin gene sequences allowing recombination with homologous chromosomal sequences has enabled us to produce, score and clone mammalian cells with the plasmid integrated into the human beta-globin locus. The planned modification was achieved in about one per thousand transformed cells whether or not the target gene was expressed.

Two recent developments suggest a route to predetermined alterations in mammalian germlines. These are, first, the characterization of mouse embryonic stem (ES) cells that can still enter the germline after genetic manipulation in culture and second, the demonstration that homologous recombination between a native target chromosomal gene and exogenous DAN can be used in culture to modify specifically the target locus. We here use gene targetting functionally to correct the mutant hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in the ES cell line which has previously been isolated and used to produce an HPRT-deficient mouse. This modification of a chosen gene in pluripotent ES cells demonstrates the feasibility of this route to manipulating mammalian genomes in predetermined ways.

Glial fibrillary acidic protein (GFAP) is a member of the family of intermediate filament structural proteins and is found predominantly in astrocytes of the central nervous system (CNS). To assess the function of GFAP, we created GFAP-null mice using gene One of the key events during astrocyte differentiation is the onset of expression of the intermediate filament glial fibrillary acidic protein (GFAP). Astrocyte precursors initially express vimentin, switching to GFAP as they mature (2, 3). Neurons make a similar switch from vimentin to the three neurofilaments during their differentiation (4). GFAP is considered a unique marker for astrocytes in the CNS, but it is also present in several other cell types in the periphery, particularly nonmyelinating Schwann cells of the peripheral nervous system (5-7). The levels of GFAP markedly increase during reactive gliosis when astrocytes undergo both hypertrophy and hyperplasia (for review see ref. 8). Recent studies of either forced overexpression or antisense inhibition of GFAP expression in cultured cells suggest a direct role for GFAP in controlling outgrowth of processes by astrocytes (9-11).Recently, two groups reported that targeted mutations in the Gfap gene do not interfere with mouse development (12,13). Preliminary studies of astrocyte numbers and morphology in these mice indicated little change, but morphology in particular has been difficult to assess in the absence of GFAP, which itself has been the standard marker for visualizing astrocytes at the light microscopic level. Upon injury, GFAPdeficient astrocytes appeared to initiate at least some of the molecular changes typical of reactive astrocytes. Given the many proposed interactions between astrocytes and neurons, changes in astrocyte structure or function might lead to changes in neuronal physiology. We have also generated GFAP-null mice using homologous recombination in embryonic stem (ES) cells. We report here that GFAP-null mice have subtle changes in astrocyte morphology, and in addition show enhanced long-term potentiation (LTP) in hippocampal neurons.

Mutations in VMD2, encoding bestrophin (best-1), cause Best vitelliform macular dystrophy (BMD), adult-onset vitelliform macular dystrophy (AVMD), and autosomal dominant vitreoretinochoroidopathy (ADVIRC). BMD is distinguished from AVMD by a diminished electrooculogram light peak (LP) in the absence of changes in the flash electroretinogram. Although the LP is thought to be generated by best-1, we find enhanced LP luminance responsiveness with normal amplitude in Vmd2 −/− mice and no differences in cellular Cl− currents in comparison to Vmd2 +/+ littermates. The putative Ca2+ sensitivity of best-1, and our recent observation that best-1 alters the kinetics of voltage-dependent Ca2+ channels (VDCC), led us to examine the role of VDCCs in the LP. Nimodipine diminished the LP, leading us to survey VDCC β-subunit mutant mice. Lethargic mice, which harbor a loss of function mutation in the β4 subunit of VDCCs, exhibited a significant shift in LP luminance response, establishing a role for Ca2+ in LP generation. When stimulated with ATP, which increases [Ca++]I, retinal pigment epithelial cells derived from Vmd2 −/− mice exhibited a fivefold greater response than Vmd2 +/+ littermates, indicating that best-1 can suppress the rise in [Ca2+]I associated with the LP. We conclude that VDCCs regulated by a β4 subunit are required to generate the LP and that best-1 antagonizes the LP luminance response potentially via its ability to modulate VDCC function. Furthermore, we suggest that the loss of vision associated with BMD is not caused by the same pathologic process as the diminished LP, but rather is caused by as yet unidentified effects of best-1 on other cellular processes.

On bipolar cells are connected to photoreceptors via a sign-inverting synapse. At this synapse, glutamate binds to a metabotropic receptor which couples to the closure of a cation-selective transduction channel. The molecular identity of both the receptor and the G protein are known, but the identity of the transduction channel has remained elusive. Here, we show that the transduction channel in mouse rod bipolar cells, a subtype of On bipolar cell, is likely to be a member of the TRP family of channels. To evoke a transduction current, the metabotropic receptor antagonist LY341495 was applied to the dendrites of cells that were bathed in a solution containing the mGluR6 agonists L-AP4 or glutamate. The transduction current was suppressed by ruthenium red and the TRPV1 antagonists capsazepine and SB-366791. Furthermore, focal application of the TRPV1 agonists capsaicin and anandamide evoked a transduction-like current. The capsaicin-evoked and endogenous transduction current displayed prominent outward rectification, a property of the TRPV1 channel. To test the possibility that the transduction channel is TRPV1, we measured rod bipolar cell function in the TRPV1 Ϫ/Ϫ mouse. The ERG b-wave, a measure of On bipolar cell function, as well as the transduction current and the response to TRPV1 agonists were normal, arguing against a role for TRPV1. However, ERG measurements from mice lacking TRPM1 receptors, another TRP channel implicated in retinal function, revealed the absence of a b-wave. Our results suggest that a TRP-like channel, possibly TRPM1, is essential for synaptic function in On bipolar cells.

The multisubunit (␣ 1S , ␣ 2 ͞␦, ␤ 1 , and ␥) skeletal muscle dihydropyridine receptor transduces transverse tubule membrane depolarization into release of Ca 2؉ from the sarcoplasmic reticulum, and also acts as an L-type Ca 2؉ channel. The ␣ 1S subunit contains the voltage sensor and channel pore, the kinetics of which are modified by the other subunits. To determine the role of the ␤ 1 subunit in channel activity and excitation-contraction coupling we have used gene targeting to inactivate the ␤ 1 gene. ␤ 1 -null mice die at birth from asphyxia. Electrical stimulation of ␤ 1 -null muscle fails to induce twitches, however, contractures are induced by caffeine. In isolated ␤ 1 -null myotubes, action potentials are normal, but fail to elicit a Ca 2؉ transient. L-type Ca 2؉ current is decreased 10-to 20-fold in the ␤ 1 -null cells compared with littermate controls. Immunohistochemistry of cultured myotubes shows that not only is the ␤ 1 subunit absent, but the amount of ␣ 1S in the membrane also is undetectable. In contrast, the ␤ 1 subunit is localized appropriately in dysgenic, mdg͞mdg, (␣ 1S -null) cells. Therefore, the ␤ 1 subunit may not only play an important role in the transport͞insertion of the ␣ 1S subunit into the membrane, but may be vital for the targeting of the muscle dihydropyridine receptor complex to the transverse tubule͞sarcoplasmic reticulum junction.

Glutamate release from photoreceptor terminals is controlled by voltage-dependent calcium channels (VDCCs). In humans, mutations in the Cacna1f gene, encoding the α 1F subunit of VDCCs, underlie the incomplete form of X-linked congenital stationary night blindness (CSNB2). These mutations impair synaptic transmission from rod and cone photoreceptors to bipolar cells. Here, we report anatomical and functional characterizations of the retina in the nob2 (no b-wave 2) mouse, a naturally occurring mutant caused by a null mutation in Cacna1f. Not surprisingly, the b-waves of both the light-and dark-adapted electroretinogram are abnormal in nob2 mice. The outer plexiform layer (OPL) is disorganized, with extension of ectopic neurites through the outer nuclear layer that originate from rod bipolar and horizontal cells, but not from hyperpolarizing bipolar cells. These ectopic neurites continue to express mGluR6, which is frequently associated with profiles that label with the

Complete congenital stationary night blindness (cCSNB) is a clinically and genetically heterogeneous group of retinal disorders characterized by nonprogressive impairment of night vision, absence of the electroretinogram (ERG) b-wave, and variable degrees of involvement of other visual functions. We report here that mutations in GPR179, encoding an orphan G protein receptor, underlie a form of autosomal-recessive cCSNB. The Gpr179(nob5/nob5) mouse model was initially discovered by the absence of the ERG b-wave, a component that reflects depolarizing bipolar cell (DBC) function. We performed genetic mapping, followed by next-generation sequencing of the critical region and detected a large transposon-like DNA insertion in Gpr179. The involvement of GPR179 in DBC function was confirmed in zebrafish and humans. Functional knockdown of gpr179 in zebrafish led to a marked reduction in the amplitude of the ERG b-wave. Candidate gene analysis of GPR179 in DNA extracted from patients with cCSNB identified GPR179-inactivating mutations in two patients. We developed an antibody against mouse GPR179, which robustly labeled DBC dendritic terminals in wild-type mice. This labeling colocalized with the expression of GRM6 and was absent in Gpr179(nob5/nob5) mutant mice. Our results demonstrate that GPR179 plays a critical role in DBC signal transduction and expands our understanding of the mechanisms that mediate normal rod vision.