RESUMO
Green Fluorescent protein (GFP) transgenic animals are accepted tools for studying various physiological processes, including organ development and cell migration. However, several in vivo studies claimed that GFP may impair transgenic animals' health. Glomerulosclerosis was observed in transgenic mice and rabbits with ubiquitous reporter protein expression. Heart-specific GFP expression evoked dilated cardiomyopathy and altered cardiac function in transgenic mouse and zebrafish lines, respectively. Moreover, growth retardation and increased axon swelling were observed in GFP and yellow fluorescent protein (YFP) transgenic mice, respectively. This review will focus on the potential drawbacks of the applications of GFP transgenic animals in biomedical research.
Assuntos
Animais Geneticamente Modificados/genética , Pesquisa Biomédica/tendências , Proteínas de Fluorescência Verde/genética , Animais , Pesquisa Biomédica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/biossíntese , Camundongos , Camundongos Transgênicos , Coelhos , Peixe-ZebraRESUMO
Cell transfer between mother and fetus were demonstrated previously in several species which possess haemochorial placenta (e.g. in humans, mice, rats, etc.). Here we report the assessment of fetal and maternal microchimerism in non-transgenic (non-TG) New Zealand white rabbits which were pregnant with transgenic (TG) fetuses and in non-TG newborns of TG does. The TG construct, including the Venus fluorophore cDNA driven by a ubiquitous cytomegalovirus enhancer, chicken ß-actin promoter (CAGGS), was previously integrated into the rabbit genome by Sleeping Beauty transposon system. Three different methods [fluorescence microscopy, flow cytometry and quantitative polymerase chain reaction (QPCR)] were employed to search for TG cells and gene products in blood and other tissues of non-TG rabbits. Venus positive peripheral blood mononuclear cells (PBMCs) were not detected in the blood of non-TG littermates or non-TG does by flow cytometry. Tissue samples (liver, kidney, skeletal and heart muscle) also proved to be Venus negative examined with fluorescence microscopy, while histology sections and PBMCs of TG rabbits showed robust Venus protein expression. In case of genomic DNA (gDNA) sourced from tissue samples of non-TG rabbits, CAGGS promoter-specific fragments could not be amplified by QPCR. Our data showed the lack of detectable cell transfer between TG and non-TG rabbits during gestation.
Assuntos
Animais Geneticamente Modificados/genética , Movimento Celular/genética , Quimerismo/embriologia , Relações Materno-Fetais , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Galinhas/genética , Elementos de DNA Transponíveis/genética , Feminino , Citometria de Fluxo , Leucócitos Mononucleares/metabolismo , Microscopia de Fluorescência , Gravidez , Regiões Promotoras Genéticas/genética , CoelhosRESUMO
Since the creation of the first transgenic rabbit thirty years ago, pronuclear microinjection remained the single applied method and resulted in numerous important rabbit models of human diseases, including cardiac deficiencies, albeit with low efficiency. For additive transgenesis a novel transposon mediated method, e.g., the Sleeping Beauty transgenesis, increased the efficiency, and its application to create cardiac disease models is expected in the near future. The targeted genome engineering nuclease family, e.g., the zink finger nuclease (ZFN), the transcription activator-like effector nuclease (TALEN) and the newest, clustered regularly interspaced short palindromic repeats (CRISPR) with the CRISPR associated effector protein (CAS), revolutionized the non-mouse transgenesis. The latest gene-targeting technology, the CRISPR/CAS system, was proven to be efficient in rabbit to create multi-gene knockout models. In the future, the number of tailor-made rabbit models produced with one of the above mentioned methods is expected to exponentially increase and to provide adequate models of heart diseases.
Assuntos
Técnicas de Transferência de Genes , Cardiopatias , Animais , Animais Geneticamente Modificados , Elementos de DNA Transponíveis/genética , Modelos Animais de Doenças , Genômica , Humanos , CoelhosRESUMO
Sepsis is a life threatening condition that arises when the body's response to an infection injures its own tissues and organs. Sepsis can lead to shock, multiple organ failure and death especially if not recognized early and treated promptly. Molecular mechanisms underlying the systemic inflammatory response syndrome associated with sepsis are still not completely defined and most therapies developed to target the acute inflammatory component of the disease are insufficient. In this study we investigated a possibility of combating sepsis in a mouse model by intravenous treatment with recombinant human tissue non-specific alkaline phosphatase (rhTNAP) derived from transgenic rabbit milk. We induced sepsis in mice by intraperitoneal injection of LPS and three hours later treated experimental group of mice by intravenous injection with rhTNAP derived from transgenic rabbits. Such treatment was proved to be physiologically effective in this model, as administration of recombinant rhTNAP successfully combated the decrease in body temperature and resulted in increased survival of mice (80 % vs. 30 % in a control group). In a control experiment, also the administration of bovine intestinal alkaline phosphatase by intravenous injection proved to be effective in increasing survival of mice treated with LPS. Altogether, present work demonstrates the redeeming effect of the recombinant tissue non-specific AP derived from milk of genetically modified rabbits in combating sepsis induced by LPS.
Assuntos
Fosfatase Alcalina/uso terapêutico , Lipopolissacarídeos/toxicidade , Sepse/induzido quimicamente , Sepse/tratamento farmacológico , Animais , Animais Geneticamente Modificados , Bovinos , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Projetos Piloto , Coelhos , Proteínas Recombinantes/uso terapêutico , Sepse/mortalidade , Taxa de Sobrevida/tendênciasRESUMO
Pluripotent stem cells have the capacity to divide indefinitely and to differentiate into all somatic cells and tissue lines. They can be genetically manipulated in vitro by knocking genes in or out, and therefore serve as an excellent tool for gene function studies and for the generation of models for some human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, many attempts have been made to generate pluripotent stem cell lines from other species. Comparative characterization of ESCs from different species would help us to understand differences and similarities in the signaling pathways involved in the maintenance of pluripotency and the initiation of differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved across different species. This report gives an overview of research into embryonic and induced pluripotent stem cells in the rabbit, an important nonrodent species with considerable merits as an animal model for specific diseases. A number of putative rabbit ESC and induced pluripotent stem cell lines have been described. All of them expressed stem cell-associated markers and maintained apparent pluripotency during multiple passages in vitro, but none have been convincingly proven to be fully pluripotent in vivo. Moreover, as in other domestic species, the markers currently used to characterize the putative rabbit ESCs are suboptimal because recent studies have revealed that they are not always specific to the pluripotent inner cell mass. Future validation of rabbit pluripotent stem cells would benefit greatly from a validated panel of molecular markers specific to pluripotent cells of the developing rabbit embryos. Using rabbit-specific pluripotency genes may improve the efficiency of somatic cell reprogramming for generating induced pluripotent stem cells and thereby overcome some of the challenges limiting the potential of this technology.
Assuntos
Células-Tronco Pluripotentes , Coelhos , Animais , Diferenciação Celular/genética , Linhagem Celular , Quimera , Células-Tronco Embrionárias/citologia , Imuno-Histoquímica/veterinária , Células-Tronco Pluripotentes Induzidas/citologia , MicroRNAs/fisiologia , Modelos Animais , Técnicas de Transferência Nuclear/veterinária , Células-Tronco Pluripotentes/citologia , Coelhos/genética , Fatores de Transcrição/fisiologiaRESUMO
The laboratory rabbit (Oryctolagus cuniculus) is widely used as a model for human diseases, because of its size, which permits non-lethal monitoring of physiological changes and similar disease characteristics. Novel transgenic tools such as, the zinc finger nuclease method and the sleeping beauty transposon mediated or BAC transgenesis were recently adapted to the laboratory rabbit and opened new opportunities in precise tissue and developmental stage specific gene expression/silencing, coupled with increased transgenic efficiencies. Many facets of human development and diseases cannot be investigated in rodents. This is especially true for early prenatal development, its long-lasting effects on health and complex disorders, and some economically important diseases such as atherosclerosis or cardiovascular diseases. The first transgenic rabbits models of arrhythmogenesis mimic human cardiac diseases much better than transgenic mice and hereby underline the importance of non-mouse models. Another emerging field is epigenetic reprogramming and pathogenic mechanisms in diabetic pregnancy, where rabbit models are indispensable. Beyond that rabbit is used for decades as major source of polyclonal antibodies and recently in monoclonal antibody production. Alteration of its genome to increase the efficiency and value of the antibodies by humanization of the immunoglobulin genes, or by increasing the expression of a special receptor (Fc receptor) that augments humoral immune response is a current demand.
Assuntos
Animais Geneticamente Modificados , Doenças Cardiovasculares , Modelos Animais de Doenças , Desenvolvimento Embrionário , Animais , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/patologia , Elementos de DNA Transponíveis/genética , Diabetes Mellitus/genética , Diabetes Mellitus/patologia , Células-Tronco Embrionárias , Técnicas de Transferência de Genes , Humanos , Camundongos , CoelhosRESUMO
Our aim was to analyse the transcription levels of the three non-classical class Ib genes SLA-6, SLA-7 and SLA-8 of the swine major histocompatibility complex in various tissues and conditions and to compare them to the transcription levels of classical class Ia genes. Twenty-five adult tissues from two pig breeds, pig renal PK15 cells infected with the Pseudorabies virus, and peripheral blood mononuclear cells (PBMCs) stimulated by lipopolysaccharide or a mixture of phorbol myristate acetate and ionomycin were included in our study. Relative transcription was quantified by quantitative real-time PCR. On average, in adult tissues and PBMCs and compared to SLA-6, the transcription level of SLA-Ia genes was 100-1000 times higher, the level of SLA-8 was 10-20 times higher, and that of SLA-7 was five times higher. Thus, SLA-8 is the most transcribed SLA-Ib gene, followed by the SLA-7 and SLA-6 genes. The highest transcription levels of SLA-Ib transcripts were found in the lymphoid organs, followed by the lung and the digestive tract. The tissue variability of expression levels was widest for the SLA-6 gene, with a 1:32 ratio between the lowest and highest levels in contrast to a 1:12 ratio for the SLA-7 and SLA-8 genes and a 1:16 ratio for the SLA-Ia genes. During PK-15 infection and PBMC stimulation, SLA-Ia and SLA-8 genes were downregulated, whereas SLA-6 and SLA-7 were upregulated, downregulated or not significantly modified. Our overall results confirm the tissue-wide transcription of the three SLA-Ib genes and suggest that they have complementary roles.
Assuntos
Antígenos de Histocompatibilidade Classe I/genética , Sus scrofa/genética , Animais , Expressão Gênica , Antígenos de Histocompatibilidade Classe I/imunologia , Leucócitos Mononucleares/imunologia , Especificidade de Órgãos , Sus scrofa/imunologia , Transcrição GênicaRESUMO
RNA processing events modulate final productivity of a given transgene. We have evaluated a series of RNA elements for their ability to enhance alpha1-antitrypsin production in mammary cells. Our results indicate the need for a case-by-case assessment of each construct design and the occurrence of gene silencing events in vivo.
Assuntos
Processamento Pós-Transcricional do RNA , RNA/metabolismo , Transgenes , Animais , Inativação Gênica , Técnicas de Transferência de Genes , Camundongos , Camundongos Transgênicos , TransfecçãoRESUMO
To achieve chondrocyte-specific deletion of floxed genes we generated a transgenic mouse line expressing the Cre recombinase under the control of the mouse type II collagen gene (Col2a1) regulatory regions. Northern and in situ hybridization analyses demonstrated the expression of the transgene (Col2a1-Cre) in cartilaginous tissues. To test the excision efficiency of Cre, the Col2a1-Cre strain was crossed with the ROSA26 reporter strain. LacZ staining of double transgenic mice revealed Cre activity in both chondrogenic and non-chondrogenic tissues. During early embryonic development (E9.5-11.5), LacZ expression was detected in tissues where the endogenous Col2a1 transcript is expressed such as the otic capsule, notochord, developing brain, sclerotome and mesenchymal condensations of future cartilage. At later stages, Cre activity was observed in all cartilaginous tissues with virtually 100% of chondrocytes being LacZ positive. These data suggest that the Col2a1-Cre mouse strain described here can be useful to achieve Cre-mediated recombination in Col2a1 expressing cells, especially in chondrocytes.
Assuntos
Colágeno/genética , Expressão Gênica , Integrases/genética , Regiões Promotoras Genéticas , Proteínas Virais , Animais , Fusão Gênica Artificial , Desenvolvimento Embrionário e Fetal , Integrases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos TransgênicosRESUMO
The rabbit kappa-casein encoding gene has previously been shown to possess two alleles. The two alleles do not differ in their coding region and in the accumulation levels of mRNA. However they differ greatly with respect to their intronic regions. The rearranged regions in the first and fourth introns were found to be inverse and complementary LINE sequences. The A allele was found to be more frequent in different European breeds. Correlation of the kappa-casein genotype with the breeding capacity in a New Zealand White rabbit stock has been examined.
Assuntos
Caseínas/genética , Polimorfismo Genético/fisiologia , Coelhos/genética , Alelos , Animais , Peso Corporal/fisiologia , Feminino , Frequência do Gene , Rearranjo Gênico , Íntrons/genética , Lactação/fisiologia , Reprodução/fisiologiaRESUMO
Transgenic mice were produced carrying the coding region of the rabbit kappa-casein gene linked to the upstream region of the rabbit whey acidic protein gene. Mice from the highest-expressing line produced 2.5 mg rabbit kappa-casein/ml in their milk. The foreign protein was associated with the casein micelles and altered micelle size, though in the high-expressing line rabbit kappa-casein also segregated into the whey fraction obtained after centrifuging the milk samples. Milk from transgenic mice had the same overall protein content as that from non-transgenic mice, except for the transgene product. However, litters fed with this transgenic mouse milk grew less well than litters given milk from non-transgenic mice. This reduction in growth was not related to changes in mammary gland structure or mammary cell morphology. Preliminary results indicated that milk from the transgenic mice had a higher viscosity.
Assuntos
Caseínas/metabolismo , Proteínas do Leite/genética , Leite/química , Animais , Northern Blotting , Caseínas/genética , Quimosina/metabolismo , Feminino , Expressão Gênica , Lactação , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/ultraestrutura , Camundongos , Camundongos Transgênicos , Micelas , Microscopia Eletrônica , Coelhos , Especificidade da Espécie , ViscosidadeRESUMO
Matrilin 1, or cartilage matrix protein, is a member of a novel family of extracellular matrix proteins. To date, four members of the family have been identified, but their biological role is unknown. Matrilin 1 and matrilin 3 are expressed in cartilage, while matrilin 2 and matrilin 4 are present in many tissues. Here we describe the generation and analysis of mice carrying a null mutation in the Crtm gene encoding matrilin 1. Anatomical and histological studies demonstrated normal development of homozygous mutant mice. Northern blot and biochemical analyses show no compensatory up-regulation of matrilin 2 or 3 in the cartilage of knockout mice. Although matrilin 1 interacts with the collagen II and aggrecan networks of cartilage, suggesting that it may play a role in cartilage tissue organization, studies of collagen extractability indicated that collagen fibril maturation and covalent cross-linking were unaffected by the absence of matrilin 1. Ultrastructural analysis did not reveal any abnormalities of matrix organization. These data suggest that matrilin 1 is not critically required for cartilage structure and function and that matrilin 1 and matrilin 3 may have functionally redundant roles.
Assuntos
Osso e Ossos/anatomia & histologia , Cartilagem/crescimento & desenvolvimento , Proteínas da Matriz Extracelular/deficiência , Glicoproteínas/deficiência , Animais , Cartilagem/química , Epífises/química , Proteínas da Matriz Extracelular/isolamento & purificação , Glicoproteínas/isolamento & purificação , Homozigoto , Imuno-Histoquímica , Proteínas Matrilinas , Camundongos , Camundongos Mutantes , Tíbia/anatomia & histologia , Distribuição Tecidual , Traqueia/químicaRESUMO
Gene-farming techniques provide an effective tool for the production of recombinant proteins in livestock. Transgenes consisting of genomic DNA sequences are as a rule more efficiently expressed than those in which the product of interest is encoded by a cDNA. However, the processing of pre-mRNA from genomic constructs may yield unexpected messenger RNAs and subsequently protein variants. We describe the appearance of different alternative mRNA splice patterns of a gene construct in which a mutant human growth hormone (hGH-N) gene is transcriptionally controlled by 2.5 kb of mouse whey acidic protein (WAP2) regulatory sequences in the mammary gland of different livestock species. Compared to the transcription products in transgenic mice harboring the same gene construct and to cell transfection experiments, expression analysis in transgenic pigs and rabbits revealed different mRNA splice patterns with regard to the proportion of the processed transcripts. Apart from already-known physiological mRNA splice products, previously undescribed processed hGH transcripts were observed in these species. Sequence analysis of the transgenes suggests that the species-specific hGH mRNA patterns may be caused by species- and tissue-specific differences in trans-acting splice factors.
Assuntos
Processamento Alternativo/genética , Glândulas Mamárias Animais/metabolismo , RNA Mensageiro/genética , Suínos/genética , Transgenes/genética , Animais , Animais Domésticos , Animais Geneticamente Modificados , Southern Blotting , Western Blotting , Análise Mutacional de DNA , Feminino , Dosagem de Genes , Hormônio do Crescimento/genética , Hormônio do Crescimento/metabolismo , Humanos , Camundongos , Proteínas do Leite/genética , Especificidade de Órgãos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/análise , Coelhos , Especificidade da Espécie , Fatores de TempoRESUMO
The transcriptional regulation of the murine gene encoding the 67-kDa form of glutamic acid decarboxylase (GAD67) was studied by beta-galactosidase histochemistry in transgenic mice carrying fusion genes between progressively longer portions of the 5'-upstream regulatory region of GAD67 and E. coli lacZ. No expression was detected in brains of mice carrying 1.3 kb of upstream sequences including a housekeeping and two conventional promoters, and two negative regulatory elements with homology to known silencers. In mice carrying the same portion of the promoter region plus the first intron, lacZ expression in the adult central nervous system was found in few, exclusively neuronal sites. The number of correctly stained GABAergic centres increased dramatically with increasing the length of the 5'-upstream region included in the construct which suggests that multiple putative spatial enhancers are located in this region. Their action is influenced by epigenetic mechanisms that may be due to site-of-integration and transgene copy-number effects. Additional cis-acting elements are needed to obtain fully correct expression in all GABAergic neurons of the adult central nervous system.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Sistema Nervoso Central/fisiologia , Proteínas de Ligação a DNA/biossíntese , Regulação da Expressão Gênica/fisiologia , Óperon Lac/genética , Fatores de Transcrição/biossíntese , Animais , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Íntrons/genética , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Fator de Transcrição CHOP , Fatores de Transcrição/genética , Transcrição Gênica/genética , beta-Galactosidase/metabolismoRESUMO
The most frequent allele of the rabbit kappa-casein (kappa-Cas)-encoding gene (A allele) has previously been shown to possess two sequences similar to those found in the 5' end of long interspersed repeated elements (LINE). Part of an inverted rabbit LINE is present in the first intron and part of a direct rabbit LINE in the fourth intron. We describe herewith a less frequent allele (B allele) that lacks both 100bp in the first intron and 1550bp in the fourth intron. It was not possible to identify any allele exhibiting only one of the deletions in a population of 55 rabbits. The 100bp present in the first intron of the A allele, but absent from the B allele, are located at the 5' end of the inverse complementary LINE and include the poly (T) track of the LINE. The 1550bp present in the fourth intron of the A allele, but absent from the B allele, include the entire direct LINE sequence. Therefore, the B allele only possesses one partial LINE sequence that is located in the first intron and is truncated when compared to the copy found in the first intron of the A allele. The B allele might thus be more recent than the A allele. Differences between the sequences of transcripts corresponding to each allele are limited to two silent mutations and three modifications in the 3' UTR. In the mammary glands of lactating rabbits, which are homozygous for both alleles, kappa-Cas mRNA accumulate to similar levels and are translated into identical kappa-Cas that are secreted at similar concentrations into milk.
Assuntos
Caseínas/genética , DNA Satélite/genética , Repetições de Microssatélites , Polimorfismo de Fragmento de Restrição , Coelhos/genética , Alelos , Animais , Sequência de Bases , Northern Blotting , DNA Complementar/genética , Desoxirribonuclease HindIII , Feminino , Genótipo , Íntrons/genética , Lactação/genética , Glândulas Mamárias Animais/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Alinhamento de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
The mouse cartilage matrix protein gene (Crtm) was isolated from a cosmid library using a mouse Crtm cDNA fragment as probe. Crtm spans 12.2 kb from the start of translation to the polyadenylation signal sequence and comprises eight exons. Sequencing of the 1.9 kb 5' flanking region revealed a TATA-like box 72 bp upstream from the initiator Met codon as well as several cis-acting motifs known to bind eukaryotic transcription factors. Analysis of the exon-intron junctions demonstrated that the last intron does not follow the gt/ag rule but belongs to the minor class of pre-mRNA introns that contain "at" and "ac" at their 5'and 3' ends, respectively. Single-strand conformation polymorphism analysis was used to map Crtm to the distal part of chromosome 4 between the microsatellite markers D4Mit16 and D4Mit339. Achodroplasia (cn), a recessive skeletal disorder in mice, has already been mapped to this region. Immunostaining for CMP and sequence of Crtm in cn/cn mice failed to reveal any disease-specific mutations, suggesting that mutations in Crtm do not cause achondroplasia.
Assuntos
Acondroplasia/genética , Cartilagem/metabolismo , Proteínas da Matriz Extracelular , Glicoproteínas/genética , Acondroplasia/patologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Cartilagem/citologia , Cruzamentos Genéticos , Éxons , Biblioteca Genômica , Glicoproteínas/biossíntese , Glicoproteínas/química , Íntrons , Fígado/metabolismo , Proteínas Matrilinas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Dados de Sequência Molecular , Polimorfismo Conformacional de Fita Simples , Mapeamento por RestriçãoRESUMO
The rabbit kappa-casein (kappa-Cas) encoding gene has been isolated as a series of overlapping DNA fragments cloned from a rabbit genomic library constructed in bacteriophage lambda EMBL3. The clones harboured the 7.5-kb gene flanked by about 2.1 kb upstream and 9 kb downstream sequences. The cloned gene is the most frequently occurring of two kappa-Cas alleles identified in New Zealand rabbits. Comparison of the corresponding domains in rabbit and bovine kappa-Cas shows that both genes comprise 5 exons and that the exon/intron boundary positions are conserved whereas the introns have diverged considerably. The first three introns are shorter in the rabbit, the second intron showing the greatest difference between the two species: 1.35 kb instead of 5.8 kb in the bovine gene. Repetitive sequence motives reminiscent of the rabbit C type repeat and the complementary inverted C type repeat were identified in the fourth and first introns, respectively. Transgenic mice were produced by microinjecting into mouse oocytes an isolated genomic DNA fragment which contained the entire kappa-Cas coding region, together with 2.1-kb 5' and 4.0-kb 3' flanking region. Expression of transgene rabbit kappa-Cas mRNA could be detected in the mammary gland of lactating transgenic mice and the production of rabbit kappa-Cas was detected in milk using species-specific antibodies. The cloned gene is thus functional.
Assuntos
Caseínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Clonagem Molecular , DNA Recombinante , Feminino , Técnicas de Transferência de Genes , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Coelhos , Análise de Sequência de DNARESUMO
A cDNA encoding the mouse cartilage matrix protein (CMP) was cloned following the reverse-transcription polymerase chain reaction and rapid amplification of cDNA ends procedures using mRNA isolated from trachea. The open reading frame encodes a product of 500 amino acids. Large parts of the protein have been completely conserved when compared to chicken and human sequences, including all 12 cysteine residues of the mature CMP. In situ hybridization reveals an even distribution of the CMP mRNA in the developing skeleton, which is followed by a zonal distribution paralleling hypertrophy and calcification. From early cartilage differentiation and onwards, CMP transcript is absent in the forming articular surfaces and intervertebral discs. Extraskeletal expression of CMP mRNA was detected in the adult eye.
Assuntos
DNA Complementar/metabolismo , Proteínas da Matriz Extracelular , Expressão Gênica , Glicoproteínas/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Osso e Ossos/metabolismo , Cartilagem/enzimologia , Cartilagem/metabolismo , Proteína de Matriz Oligomérica de Cartilagem , Galinhas , Clonagem Molecular , Sequência Conservada , DNA Complementar/biossíntese , DNA Complementar/química , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Embrião não Mamífero , Glicoproteínas/análise , Glicoproteínas/genética , Humanos , Hibridização In Situ , Proteínas Matrilinas , Camundongos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Fases de Leitura Aberta , Especificidade de Órgãos , Reação em Cadeia da Polimerase , Sondas RNA , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Homologia de Sequência de Aminoácidos , Traqueia/metabolismo , Transcrição GênicaRESUMO
Genetic analysis of RFLP was used for detection of genotype and allele frequencies of kappa-casein in Slovakian Spotted (60 bulls) and Slovakian Pinzgau (22 bulls) cattle breeds, according to the method of Medrano et al. (1990). DNA was prepared from the semen of animals. In the Slovakian Spotted breed the frequencies of alleles were as follows: kappa-CnA = 0.666, kappa-CnB = 0.333. The frequencies of kappa-Cn A/A, A/B and B/B genotypes were 45.00, 43.33 and 11.66, respectively. In the 22 tested Pinzgau bulls, the frequencies of the A and B alleles were 0.682 and 0.318, respectively. The percentual occurrence of genotypes was also determined: 54.54 (kappa-Cn A/A), 27.27 (kappa-Cn A/B) and 18.18 (kappa-Cn B/B). Comparing our own results with those of Mácha et al. (1968), who carried out the analysis of distribution of the kappa-casein genetic variants in the same cattle breeds by starch gel electrophoresis of the milk samples of 170 cows (Tab. I), the 16 p.c. decrease of the allele B in the Slovakian Spotted cattle, lasting about 30 years, is very remarkable. The occurrence of homozygous genotype BB decreased by 35 p.c. In addition, the homozygous genotype AA increased by about 18 p.c. and the occurrence of heterozygous genotype is also higher by nearly 17 p.c. In the same comparison of the Slovakian Pinzgau breed, no difference was estimated in the allele frequencies of kappa-Cn (Tab. I).(ABSTRACT TRUNCATED AT 250 WORDS)