RESUMO
We have developed a novel method of DNA extraction combined with a high-throughput method of gene detection allowing thousands of potentially transgenic chicks to be screened quickly and reliably. By using this method and a replication-deficient retroviral vector based on avian leukosis virus (ALV), we have demonstrated germline transmission of three different transgenes. Several generations of chickens carrying intact transgenes were produced, validating the use of the ALV retroviral vectors for large-scale production of transgenic flocks. Fourth-generation chicks that were nontransgenic, hemizygous, or homozygous for the transgene were identified with the combined genetic screening methods.
Assuntos
Animais Geneticamente Modificados , Galinhas/genética , Vetores Genéticos , Retroviridae/genética , Replicação Viral , Alelos , Animais , Southern Blotting , Cloranfenicol O-Acetiltransferase/genética , DNA/sangue , Desoxirribonuclease HindIII , Resistência a Medicamentos/genética , Homozigoto , Masculino , Neomicina , Ovalbumina/genética , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Transgenes/genéticaRESUMO
Effective transcript profiling in animal systems requires isolation of homogenous tissue or cells followed by faithful mRNA amplification. Linear amplification based on cDNA synthesis and in vitro transcription is reported to maintain representation of mRNA levels, however, quantitative data demonstrating this as well as a description of inherent limitations is lacking. We show that published protocols produce a template-independent product in addition to amplifying real target mRNA thus reducing the specific activity of the final product. We describe a modified amplification protocol that minimizes the generation of template-independent product and can therefore generate the desired microgram quantities of message-derived material from 100 ng of total RNA. Application of a second, nested round of cDNA synthesis and in vitro transcription reduces the required starting material to 2 ng of total RNA. Quantitative analysis of these products on Caenorhabditis elegans Affymetrix GeneChips shows that this amplification does not reduce overall sensitivity and has only minor effects on fidelity.
Assuntos
Perfilação da Expressão Gênica , RNA Mensageiro/metabolismo , Animais , Caenorhabditis elegans/genética , DNA Complementar/genética , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
There are seven conserved motifs (IA, IB, and II to VI) in DNA helicase II of Escherichia coli that have high homology among a large family of proteins involved in DNA metabolism. To address the functional importance of motifs II to VI, we employed site-directed mutagenesis to replace the charged amino acid residues in each motif with alanines. Cells carrying these mutant alleles exhibited higher UV and methyl methanesulfonate sensitivity, increased rates of spontaneous mutagenesis, and elevated levels of homologous recombination, indicating defects in both the excision repair and mismatch repair pathways. In addition, we also changed the highly conserved tyrosine(600) in motif VI to phenylalanine (uvrD309, Y600F). This mutant displayed a moderate increase in UV sensitivity but a decrease in spontaneous mutation rate, suggesting that DNA helicase II may have different functions in the two DNA repair pathways. Furthermore, a mutation in domain IV (uvrD307, R284A) significantly reduced the viability of some E. coli K-12 strains at 30 degrees C but not at 37 degrees C. The implications of these observations are discussed.