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Microphthalmia-Associated Transcription Family Translocation Renal Cell Cancer
Published in Dongyou Liu, Handbook of Tumor Syndromes, 2020
Specific diagnosis of MIT family tRCC relies on the use of (i) immunohistochemistry, (ii) break-apart fluorescence in situ hybridization (FISH), and (iii) reverse transcriptase-polymerase chain reaction (RT-PCR)/5′-rapid amplification of cDNA ends (5′-RACE)/karyotyping. Whereas FFPE specimens are applicable to the first two approaches, fresh specimens are necessary for the third approach [37].
The Immunoglobulin Variable-Region Gene Repertoire and Its Analysis
Published in Cliburn Chan, Michael G. Hudgens, Shein-Chung Chow, Quantitative Methods for HIV/AIDS Research, 2017
Thomas B. Kepler, Kaitlin Sawatzki
Because of the large number of different V genes, one can multiplex pools of 5′ primers that together cover the V genes, or one may use 5′ RACE (rapid amplification of cDNA ends), which adds a known template switch primer to the 5′ end of mRNA during cDNA synthesis by template-switching reverse transcriptase. In either case, one would use a more modest multiplexed pool of 3′ primers to the constant-region genes.
The Evolution of MAbs from Research Reagents to Mainstream Commercial Therapeutics
Published in Maurizio Zanetti, J. Donald Capra, The Antibodies, 1999
Efficient expression vectors have been developed to take advantage of RACE-PCR (rapid amplification of cDNA ends) technology that can amplify cDNA produced from mRNA transcripts rapidly and reproducibly PCR primers containing restriction sites, designed to retain the natural consensus amino acid sequence of the antibody, allow rapid insertion of variable region genes into a vector containing constant region genes of the desired isotype. Poor choice of restriction sites can result in the substitution of an “unnatural” amino acid at the junction point with potentially immunogenic consequences. The vectors we use at IDEC incorporate immunoglobulin genes in a tandem arrangement, which reduces the size of the plasmid, requires only a single selectable marker, and leads to better stoichiometry between heavy- and light-chain production.
Pleiotropy of the Drosophila melanogaster foraging gene on larval feeding-related traits
Published in Journal of Neurogenetics, 2018
A. M. Allen, I. Anreiter, A. Vesterberg, S. J. Douglas, M. B. Sokolowski
Allen et al. (2017) experimentally defined foraging’s transcription start sites, termination sites, and splicing patterns utilizing rapid amplification of cDNA ends (RACE) and full-length cDNA sequencing. As mentioned above, this uncovered four independent promoters pr1–4, that produce 21 transcripts with nine distinct open reading frames. The authors postulated that the use of alternative promoters and splicing at this locus can generate diversity and flexibility in the regulation of gene expression and function. They also generated a null allele (for0) using homologous recombination to precisely delete foraging; recombineering was used to reintegrate a full genomic copy into the genome in increasing doses of the gene to rescue the (for0) phenotypes. They found that a total loss of foraging expression in larvae resulted in reduced larval foraging path length, reduced food intake, and an increase in triglyceride levels (Allen et al., 2017). Their results proved that these larval phenotypes were influenced by foraging and suggested that they may be independently regulated from within the locus (Allen et al., 2017).
Porcine interleukin-6 enhances the expression of CYP2C33 through a constitutive androstane receptor/retinoid X receptor-mediated pathway
Published in Xenobiotica, 2019
Lixia Xie, Yucheng He, Xiaoqiao Zhou, Xiaowen Li, Xiue Jin, Xiliang Wang, Deshi Shi
Using SuperScript III Reverse Transcriptase, DNA-free total RNA was reverse transcribed to produce cDNA using a modified oligo-dT for 3′ rapid amplification of cDNA ends (RACE) and gene-specific primers for RXRα for 5′ RACE. To generate 5′-modified cDNA, cDNA from mRNA templates was subjected to template switching using a template-switching oligo with SuperScript III Reverse. Transcriptase at 42 °C for 90 min. PCR was then used to amplify the 3′ ends of RXRα cDNA with a universal primer mix (UPM) and gene-specific primers for RXRα using PrimeSTAR HS DNA Polymerase. The 5′ ends of RXRα cDNA were also amplified using UPM and gene-specific primers for RXRα. PCR products were analysed by agarose gel electrophoresis and sequenced.
NAb-seq: an accurate, rapid, and cost-effective method for antibody long-read sequencing in hybridoma cell lines and single B cells
Published in mAbs, 2022
Hema Preethi Subas Satish, Kathleen Zeglinski, Rachel T. Uren, Stephen L. Nutt, Matthew E. Ritchie, Quentin Gouil, Ruth M. Kluck
Conventional sequencing of antibody genes from hybridoma cell lines involves PCR amplification of antibody variable regions (VH and VL) followed by low-throughput and partial-length Sanger sequencing. Primer sets for antibody gene amplification must account for variability at the 5’ (leader sequence of framework region 1 (FR1)) and 3’ end (FR4, hinge, or constant region) on the transcripts. This requires the intricate design of many primer pairs and/or degenerate primers. Although they do not guarantee unbiased amplification, validated primer sets are available for mouse and human loci. In other species, however, the lack of such primer sets limits conventional antibody sequencing. The use of 5’ RACE (Rapid Amplification of cDNA ends), or the more recent but conceptually equivalent template-switching, halves the complexity, as it only requires primers specific to the 3’ ends of the transcripts (FR4, hinge, or constant region). This technique has been successfully applied to sequence rat, mouse, and human antibody transcripts,9–11 including from single B cells.12 In addition to these complexities of variable region PCR, Sanger sequencing by commercial providers is costly and slow: around US$800 (2 weeks) for the variable regions (VH and VL) and US$2,000 (4 weeks) for the full-length antibody (variable and constant regions). Even when performing Sanger sequencing in-house, there is still an estimated turnaround time of 5 days and cost of US$70–120 per antibody.10 Sanger sequencing is further complicated by the potential presence of multiple heavy- and light-chain transcripts in the same cell. An estimated 30% of all hybridomas express more than one productive heavy or light chain.13