By Shondra M. Pruett-Miller (eds.)
This new version explores present and rising mutagenesis equipment focusing particularly on mammalian platforms and well-known version organisms via accomplished insurance and special protocols. because the first variation, significant advances and discoveries have made chromosomal mutagenesis a regularly occurring method and one who is out there to any molecular biology laboratory, and this assortment presents designated protocols, case-studies, and experiences from thought-leaders within the box. Written within the hugely profitable Methods in Molecular Biology sequence structure, chapters comprise introductions to their respective subject matters, lists of the required fabrics and reagents, step by step, without problems reproducible laboratory protocols, and tips about troubleshooting and heading off identified pitfalls.
Authoritative and completely up to date, Chromosomal Mutagenesis, moment Edition goals to assist pace medical discovery and relief within the subsequent advances within the field.
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If pSNL feeders are used, the puromycin concentration should be 3 μg/ml. 11. , for a library of 1,000 mutants, freeze in aliquots of at least 20,000 cells). When the frozen cells are thawed, it is important to measure the viability and estimate how many viable cells are in the initial plating. A low viable cell count (less than three times the library complexity) risks distorting the representation of mutants in the pool and losing mutants. 12. It can be useful to routinely fix samples as cells are passaged to monitor the culture.
Even with efficient insertional mutagens like DNA transposons, the diploid nature of the mammalian genome has always hampered loss-of-function screening. In the cell culture system, it is in principle impossible to convert randomly generated genome-wide heterozygous mutations into homozygotes. This issue was partially solved by increasing the frequency of loss-of-heterozygosity (LOH) using a Blm RecQ DNA helicase deficient background [34, 35]. However, since the frequency of LOH is only 10−4 events per cell per generation [34, 36], homozygous cells represent a very small fraction of a population consisting of at least 10,000 times more heterozygous mutants.
Resuspend 1 × 107 cells in 800 μl PBS and mix them with 1 μg transposon vector and 10 μg pCMV-hyPBase. Transfer the 24 Stephen J. Pettitt et al. mixture to an electroporation cuvette. Electroporate using Bio-Rad GenePulser (270 V, 500 μF) (see Note 9). 5. Plate cells on three 10-cm feeder plates. Plating 1/100 and 1/1,000 of the volume on six-well plates is also useful to estimate the number of mutants later (see step 7). 6. On the following day, replace the medium with ES cell medium containing 1 μg/ml puromycin.