Cloning assays

Fig. 21. Distribution of EH activities from 3500 and 4600 clones assayed from the 2.5mM ( )...

Deldar, A., Lewis, H., Bloom, J. and Weiss, L. (1988) Reproducibile cloning assays for in vitro growth of canine hematopoietic progenitor cells and their potential applications in investigative hemato-toxicity. American Journal of Veterinary Research, 49, 1393-1401. 

Tates, A.D., van Dam, F.J., de Zwart, F.A., van Teylingen, C.M.M. Natarajan, A.T. (1994) Development of a cloning assay with high cloning efficiency to detect induction of 6-thio-guanine-resistant lymphocytes in spleen of adult mice following in vivo inhalation exposure to 1,3-butadiene. Mutat. Res., 309, 299-306... 

Fig. 2 An LB agar plate showing the result of a Topo Ta Cloning assay for allelic cloning separation...

The trichothecene mycotoxins are cytotoxic to most eukaryotic cells.46 A number of cytotoxicity assays have been developed and include survival and cloning assays, measuring protein and deoxyribonucleic acid (DNA) synthesis by radiolabeling procedures, and a neutral red cell-viability assay. A minimum of 24 to 48 hours is required to measure the effects of the trichothecene mycotoxins on cell viability. 

Salmon SE, Hersh EM. Sensitivity of multiple myeloma to imexon in the human tumor cloning assay. J Natl Cancer Inst 1994 86(3) 228-30. 

For measurement of sister chromatid exchanges, cells were grown for two cycles in medium containing 10 lM bromodeoxyuridine. Differentiation of sister chromatids was achieved by the method of Goto et al, (5). At least 20 metaphases were analyzed to determine sister chromatid exchanges for each clone. Assays for protein and DNA were performed as previously described (6,7). 

In 1975, the first successful production of MAbs was reported (44). By fusing normal antibody-producing cells with a B-ceU tumor (myeloma), hybridoma cell lines resulted which produced antibodies having a specificity to only one deterrninant on an antigen ie, all the antibodies produced from the cell line are identical. These studies resulted in a standard approach to MAb production. In this approach, the hybridoma cells are produced in large quantities in culture and screened to select specific clones producing the desired MAb using an appropriate assay. The selected clones are then expanded in culture (or in animals), the cells are collected, and the MAbs are extracted and purified. 

Expression vectors are engineered so that any cloned insert can be transcribed into RNA, and, in many instances, even translated into protein. cDNA expression libraries can be constructed in specially designed vectors derived from either plasmids or bacteriophage A. Proteins encoded by the various cDNA clones within such expression libraries can be synthesized in the host cells, and if suitable assays are available to identify a particular protein, its corresponding cDNA clone can be identified and isolated. Expression vectors designed for RNA expression or protein expression, or both, are available. 

Acantbephyra, 162, 336 Acantboscina, 336 Acholoe, 335 Achromobacter, 35, 36 Acorn worms (enteropneusts), 315 Acylhomoserine lactone, 43 Advice to students, 375 Aequorea, 159, 161, 162, 334, 375 Aequorea aequorea, 92-94, 346 collection, 93, 94 distribution, 92 squeezate, 94 synonyms, 92 Aequorea GFP, 150-154 chromophore, 153 cloning, 154 crystallization, 130 fluorescence quantum yield, 152 isolation, 129 molecular weight, 152 spectral properties, 130, 152 Aequorea victoria, 92 Aequorin, 92-129, 159, 160,172,173, 175, 346, 349, 350, 364, 375 assay, 98... 

Bacterial bioluminescence, 30-46 factors required, 31 general scheme, 32 in vivo luminescence, 41 luminescence reaction, 37, 38 Bacterial luciferase, 33-35, 343 assay, 39 cloning, 34 crystal structure, 34 extraction and purification, 34 inactivation, 34, 35 molecular weight, 34 properties, 34 storage, 35 subunits, 34... 

When assayed in HEK293 cells transfected with the cloned human, rat and guinea pig TRPVl, (23a) showed similar potencies. Not unexpeetedly, (23a) showed poor metabolic stability and a structure-activity study to optimize potency and drug-like properties was initiated. Modification on the left-handed A -aryl section showed that ... 

Any of a number of plates containing a series of wells in which to store reagents, clones, etc., or perform individual assays. Typically, these plates are constructed of a variety of clear and opaque plastics, and contain 96, 384, or 1,536 individual wells, although 24-well and 3,456-well plates are also available. 

The standard RNAs were used to test the ability of the bDNA assay to quantify accurately target RNAs regardless of size or slight sequence variation. Standard RNA preparations of 1.3,2.2, and 3.2 kb showed no detectable effect on quantitation. The quantitation of standard transcripts prepared from clones of HCV sub-type la and 3a differed by a factor of 1.6-fold with one probe design and were indistinguishable with another probe design. These two 475-mer transcripts differed at 30 positions. 

Figure 5.2. High-throughput mating assay for two-hybrid protein interaction screening. Yeast strains containing individual bait and prey clones are combined in a well and allowed to mate. Diploids are then selected and scored for a protein-protein interaction using the selection provided by the transcriptional reporter gene.

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