RESUMEN
Transgenic animals incorporating human antibody genes are extremely attractive for drug development because they obviate subsequent antibody humanization procedures required for therapeutic translation. Transgenic platforms have previously been established using mice, but also more recently rats, chickens, and cows and are now in abundant use for drug development. However, rabbit-based antibody generation, with a strong track record for specificity and affinity, is able to include gene conversion mediated sequence diversification, thereby enhancing binder maturation and improving the variance/selection of output antibodies in a different way than in rodents. Since it additionally frequently permits good binder generation against antigens that are only weakly immunogenic in other organisms, it is a highly interesting species for therapeutic antibody generation. We report here on the generation, utilization, and analysis of the first transgenic rabbit strain for human antibody production. Through the knockout of endogenous IgM genes and the introduction of human immunoglobulin sequences, this rabbit strain has been engineered to generate a highly diverse human IgG antibody repertoire. We further incorporated human CD79a/b and Bcl2 (B-cell lymphoma 2) genes, which enhance B-cell receptor expression and B-cell survival. Following immunization against the angiogenic factor BMP9 (Bone Morphogenetic Proteins 9), we were able to isolate a set of exquisitely affine and specific neutralizing antibodies from these rabbits. Sequence analysis of these binders revealed that both somatic hypermutation and gene conversion are fully operational in this strain, without compromising the very high degree of humanness. This powerful new transgenic strategy will allow further expansion of the use of endogenous immune mechanisms in drug development.
Asunto(s)
Animales Modificados Genéticamente , Afinidad de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Linfocitos B/inmunología , Inmunoglobulina G/inmunología , Animales , Humanos , Inmunoglobulina G/genética , ConejosRESUMEN
We have developed a robust platform to generate and functionally characterize rabbit-derived antibodies using B cells from peripheral blood. The rapid high throughput procedure generates a diverse set of antibodies, yet requires only few animals to be immunized without the need to sacrifice them. The workflow includes (i) the identification and isolation of single B cells from rabbit blood expressing IgG antibodies, (ii) an elaborate short term B-cell cultivation to produce sufficient monoclonal antigen specific IgG for comprehensive phenotype screens, (iii) the isolation of VH and VL coding regions via PCR from B-cell clones producing antigen specific and functional antibodies followed by the sequence determination, and (iv) the recombinant expression and purification of IgG antibodies. The fully integrated and to a large degree automated platform (demonstrated in this paper using IL1RL1 immunized rabbits) yielded clonal and very diverse IL1RL1-specific and functional IL1RL1-inhibiting rabbit antibodies. These functional IgGs from individual animals were obtained at a short time range after immunization and could be identified already during primary screening, thus substantially lowering the workload for the subsequent B-cell PCR workflow. Early availability of sequence information permits one to select early-on function- and sequence-diverse antibodies for further characterization. In summary, this powerful technology platform has proven to be an efficient and robust method for the rapid generation of antigen specific and functional monoclonal rabbit antibodies without sacrificing the immunized animal.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/sangre , Linfocitos B/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/sangre , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos/inmunología , Separación Celular , Células Cultivadas , Células Clonales , Epítopos/inmunología , Células HEK293 , Humanos , Inmunización , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/inmunología , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-33 , Interleucinas/metabolismo , Unión Proteica , Conejos , Receptores de Superficie Celular/metabolismoRESUMEN
Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs) introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM) locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM(+) and IgG(+) B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with â¼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.
Asunto(s)
Desoxirribonucleasas/química , Desoxirribonucleasas/genética , Inmunoglobulina M/genética , Ingeniería de Proteínas/métodos , Dedos de Zinc , Alelos , Animales , Secuencia de Bases , Exones/genética , Femenino , Técnicas de Inactivación de Genes , Sitios Genéticos/genética , Humanos , Inmunoglobulina G/genética , Inmunoglobulina M/deficiencia , Masculino , Microinyecciones , Datos de Secuencia Molecular , Mutación/genética , Oocitos/metabolismo , ARN Mensajero/genética , Conejos , Reproducibilidad de los ResultadosRESUMEN
Neurons express multiple types of voltage-gated calcium (Ca2+) channels. Two subtypes of neuronal L-type Ca2+ channels are encoded by CaV1.2 and CaV1.3 pore-forming subunits. To compare targeting of CaV1.2 and CaV1.3 L-type Ca2+ channels, we transfected rat hippocampal neuronal cultures with surface-epitope-tagged sHA-CaV1.2 or sHA-CaV1.3a constructs and found that: (i) both sHA-CaV1.2 and sHA-CaV1.3a form clusters on the neuronal plasma membrane surface; (ii) when compared with sHA-CaV1.2 surface clusters, the sHA-CaV1.3a surface clusters were 10% larger and 25% brighter, but 35% less abundant; (iii) 81% of sHA-CaV1.2 surface clusters, but only 48% of sHA-CaV1.3a surface clusters, co-localized with synapsin clusters; (iv) co-expression with GFP-Shank-1B had no significant effect on sHA-CaV1.2 surface clusters, but promoted formation and synaptic localization of sHA-CaV1.3a surface clusters. In experiments with dihydropyridine-resistant CaV1.2 and CaV1.3a mutants we demonstrated that CaV1.3a L-type Ca2+ channels preferentially mediate nuclear pCREB signaling in hippocampal neurons at low, but not at high, levels of stimulation. In experiments with primary neuronal cultures from CaV1.3 knockout mice we discovered that CaV1.3 channels play a more important role in pCREB signaling in striatal medium spiny neurons than in hippocampal neurons. Our results provide novel insights into the function of CaV1.2 and CaV1.3 L-type Ca2+ channels in the brain.
Asunto(s)
Proteína de Unión a CREB/metabolismo , Canales de Calcio Tipo L/fisiología , Canales de Calcio/fisiología , Neuronas/metabolismo , Animales , Calcio/fisiología , Canales de Calcio/química , Canales de Calcio Tipo L/química , Células Cultivadas , Embrión de Mamíferos , Embrión no Mamífero , Expresión Génica/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/citología , Inmunohistoquímica/métodos , Ratones , Ratones Noqueados , Modelos Moleculares , Mutagénesis/fisiología , Neuronas/efectos de los fármacos , Oocitos , Estructura Terciaria de Proteína/fisiología , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Sinapsinas/metabolismo , XenopusRESUMEN
Interactions of the II-III loop of the voltage-gated Ca(2+) channel alpha(1S) subunit with the Ca(2+) release channel (RyR1) are essential for skeletal-type excitation-contraction (EC) coupling. Here, we characterized the binding site of the monoclonal alpha(1S) antibody mAB 1A and used it to probe the structure of the II-III loop in chimeras with different EC coupling properties. Phage-display epitope mapping of mAB 1A revealed a minimal consensus binding sequence X-P-X-X-D-X-P. Immunofluorescence labeling of (1S), alpha(1C), alpha(1D), and of II-III loop chimeras expressed in dysgenic myotubes established that mAB 1A reacted specifically with amino acids 737-744 in the II-III loop of alpha(1S), which is within the domain (D734-L764) critical for bidirectional coupling with RyR1. Comparing mAB 1A immunoreactivity with known structural and functional properties of II-III loop chimeras in which the non-conserved skeletal residues were systematically mutated to their cardiac counterparts indicated a correlation of mAB 1A immunoreactivity and skeletal-type EC coupling.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Sitios de Unión de Anticuerpos , Canales de Calcio/metabolismo , Músculo Esquelético/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Bacteriófago M13/genética , Canales de Calcio/química , Línea Celular , Epítopos/genética , Epítopos/inmunología , Homología de Secuencia de AminoácidoRESUMEN
A bacterial artificial chromosome (BAC) library was created using partially digested rabbit chromosomal DNA. Four BAC clones spanning about 0.5 Mb of the rabbit immunoglobulin (Ig) heavy chain locus were isolated and sequenced. Three of the BAC clones were partially overlapping. Thirty-four V elements, 11 D elements, DQ52, six J elements and the coding regions of Cmicro, Cgamma, C and four Calpha genes were identified and characterized. Additionally, the sequence of a fosmid clone spanning Calpha13 and 30 kb 3'enhancer region was determined. The organization of the locus and the potential function of newly identified functional and structural elements are discussed.
Asunto(s)
Cadenas Pesadas de Inmunoglobulina/genética , Conejos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromosomas Artificiales Bacterianos/genética , ADN/química , ADN/genética , Biblioteca Genómica , Mutación de Línea Germinal , Inmunoglobulina E/genética , Masculino , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Outer hair cells (OHC) serve as electromechanical amplifiers that guarantee the unique sensitivity and frequency selectivity of the mammalian cochlea. It is unknown whether the afferent fibres connected to adult OHCs are functional. If so, voltage-activated Ca2+ channels would be required for afferent synaptic transmission. In neonatal OHCs, Ca2+ channels seem to play a role in maturation since OHCs from Cav1.3-deficient (Cav1.3-/-) mice degenerate shortly after the onset of hearing. We therefore studied whole-cell Ca2+ currents in outer hair cells aged between postnatal day 1 (P1) and P8. OHCs showed a rapidly activating inward current that was 1.8 times larger with 10 mM Ba2+ as charge carrier (IBa) than with equimolar Ca2+ (ICa). IBa started activating at -50 mV with Vmax = -1.9 +/- 6.9 mV, V0.5 = -15.0 +/- 7.1 mV and k = 8.2 +/- 1.1 mV (n = 34). The peak IBa showed negligible inactivation (3.6 % after 300 ms) whereas the ICa (10 mM Ca2+) was inactivated by 50.7 %. OHC IBa was reduced by 33.5 +/- 10.3 % (n = 14) with 10 microM nifedipine and increased to 178.5 +/- 57.8 % (n = 14) by 5 microM Bay K 8644. A dose-response curve for nifedipine revealed an IC50 of 2.3 microM, a Hill coefficient of 2.7 and a maximum block of 36 %. Average IBa density in OHCs was 24.4 +/- 10.8 pA pF-1 (n = 105) which is only 38 % of the value in inner hair cells. Single cell RT-PCR revealed expression of Cav1.3 in OHCs. In OHCs from Cav1.3-/- mice, Ba2+ current density was reduced to 0.6 +/- 0.5 pA pF-1 (n = 9) indicating that > 97 % of the Ca2+ channel current in OHCs flows through Cav1.3.
Asunto(s)
Canales de Calcio Tipo L/fisiología , Señalización del Calcio/fisiología , Células Ciliadas Auditivas Externas/fisiología , Animales , Animales Recién Nacidos , Bario/farmacología , Calcio/farmacología , Células Ciliadas Auditivas Internas/fisiología , Cinética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Ratones EndogámicosRESUMEN
The spontaneous activity of pacemaker cells in the sino-atrial node (SAN) controls the heart rhythm and rate under physiological conditions. Pacemaker activity in SAN cells is due to the presence of the diastolic depolarization, a slow depolarization phase that drives the membrane voltage from the end of an action potential to the threshold of a new action potential. SAN cells express a wide array of ionic channels, but we have limited knowledge about their functional role in pacemaker activity and we still do not know which channels play a prominent role in the generation of the diastolic depolarization. It is thus important to provide genetic evidence linking the activity of genes coding for ionic channels to specific alterations of pacemaker activity of SAN cells. Here, we show that target inactivation of the gene coding for alpha(1D) (Ca(v)1.3) Ca(2+) channels in the mouse not only significantly slows pacemaker activity but also promotes spontaneous arrhythmia in SAN pacemaker cells. These alterations of pacemaker activity are linked to abolition of the major component of the L-type current (I(Ca,L)) activating at negative voltages. Pharmacological analysis of I(Ca,L) demonstrates that Ca(v)1.3 gene inactivation specifically abolishes I(Ca,L) in the voltage range corresponding to the diastolic depolarization. Taken together, our data demonstrate that Ca(v)1.3 channels play a major role in the generation of cardiac pacemaker activity by contributing to diastolic depolarization in SAN pacemaker cells.
Asunto(s)
Canales de Calcio Tipo L/fisiología , Nodo Sinoatrial/fisiología , Potenciales de Acción , Adulto , Animales , Secuencia de Bases , Canales de Calcio Tipo L/genética , Cartilla de ADN , Humanos , Ratones , Ratones Noqueados , Persona de Mediana Edad , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
There is growing evidence linking alterations in serotonergic signaling in the prefrontal cortex to the etiology of schizophrenia. Prefrontal pyramidal neurons are richly innervated by serotonergic fibers and express high levels of serotonergic 5-HT(2)-class receptors. It is unclear, however, how activation of these receptors modulates cellular activity. To help fill this gap, whole cell voltage-clamp and single-cell RT-PCR studies of acutely isolated layer V-VI prefrontal pyramidal neurons were undertaken. The vast majority (>80%) of these neurons had detectable levels of 5-HT(2A) or 5-HT(2C) receptor mRNA. Bath application of 5-HT(2) agonists inhibited voltage-dependent Ca(2+) channel currents. L-type Ca(2+) channels were a particularly prominent target of this signaling pathway. The L-type channel modulation was blocked by disruption of G(alphaq) signaling or by inhibition of phospholipase Cbeta. Antagonism of intracellular inositol trisphosphate signaling, chelation of intracellular Ca(2+), or depletion of intracellular Ca(2+) stores also blocked this modulation. Inhibition of the Ca(2+)-dependent phosphatase calcineurin prevented receptor-mediated modulation of L-type currents. Last, the 5-HT(2) receptor modulation was robustly expressed in neurons from Ca(v)1.3 knockout mice. These findings argue that 5-HT(2) receptors couple through G(alphaq) proteins to trigger a phospholipase Cbeta/inositol trisphosphate signaling cascade resulting in the mobilization of intracellular Ca(2+), activation of calcineurin, and inhibition of Ca(v)1.2 L-type Ca(2+) currents. This modulation and its blockade by atypical neuroleptics could have wide-ranging effects on synaptic integration and long-term gene expression in deep-layer prefrontal pyramidal neurons.
Asunto(s)
Calcineurina/metabolismo , Canales de Calcio Tipo L/metabolismo , Isoenzimas/metabolismo , Corteza Prefrontal/fisiología , Células Piramidales/fisiología , Receptores de Serotonina/metabolismo , Fosfolipasas de Tipo C/metabolismo , Animales , Calcio/metabolismo , Canales de Calcio Tipo N/metabolismo , Canales de Calcio Tipo R/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Expresión Génica/fisiología , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Fosfatos de Inositol/metabolismo , Isoenzimas/genética , Técnicas de Placa-Clamp , Fosfolipasa C beta , Corteza Prefrontal/citología , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Receptor de Serotonina 5-HT2A , Receptor de Serotonina 5-HT2B , Receptores de Serotonina/genética , Transducción de Señal/fisiología , Fosfolipasas de Tipo C/genéticaRESUMEN
Adult inner hair cells (IHCs) possess voltage-activated Ca2+ currents that couple receptor potentials to transmitter release at the afferent synapses. Before the onset of hearing both IHCs and outer hair cells (OHCs) exhibit Ca2+ currents. More than 90% of neonatal hair cell (HC) currents flow through alpha1D Ca2+ channel subunits because they are absent in both IHCs and OHCs from alpha1D-/- mice and residual currents are insensitive to L-type agonists. Since lack of the alpha1D-subunit leads to HC degeneration and profound deafness, class D L-type Ca2+ currents seem to be crucial for the development and functioning of the inner ear. Neonatal HC Ca2+ currents were studied using the whole-cell patch clamp technique. They showed rapid activation, rapid deactivation and very little inactivation. They started activating as negative as -65mV. In contrast to alpha1C-mediated (classical L-type) Ca2+ currents, they showed a rather low sensitivity to various L-type antagonists. 10 microM nifedipine e.g. blocked HC Ca2+ currents by about 40% whereas class C L-type Ca2+ currents are completely blocked by 100nM nifedipine. The L-type channel agonist Bay K 8644 increased the HC Ca2+ current by 100-200% and shifted the IV curve to more negative potentials which is similar to its effects in alpha1C-mediated Ca2+ currents.