The materials used in the study were monoclonal anti-myosin (skeletal, fast) clone My-32 (Sigma, Madrid, Spain), monoclonal anti-myosin (skeletal, slow) clone NOQ7.5.4D (Sigma), agarose (Sigma), protein A (Sigma) and loading buffer Nupage LDS sample buffer (Novex, California, USA).
Subcloning of slow clones gave rise to a mixture of slow-growing and fast-growing colonies [ rho0 (s), L1993], whereas subcloning of fast clones only yielded fast-growing ones [ rho0 (f), L1994].
The proteins in Group A were DE between the fast and slow clones, their respective mRNAs were expressed above the microarray detection threshold but no change in mRNA expression was observed.
Looking at the chromatin-dynamics parameter set that best fit the flow-cytometry distributions for all clones, our study revealed that for the six clones, mean burst sizes were between 30.0 and 118.9 mRNAs per burst, and mean closed times between 756.7 minutes (~12 hours) for the fastest clone to 5197.6 minutes (~3.5 days) for the slowest clone.
In fact, if we had not found in our cell-free experiments with linear DNA that they had the potential to work in cells, we would not have attempted to implement them in E. coli and carried on when toxicity and loading effects further slowed cloning and in vivo testing.
Being too slow to clone has in fact hurt Xiaonei, which is now being overshadowed by Kaixin001's 90% month-to-month growth rate.
Protein lysates were run on SDS-PAGE gels, transferred to PVDF membranes, and probed with antibodies to Sox6 (Abcam54, Abcam), slow myosin (clone NOQ7.5.4D, Sigma), SRF (sc-335, Santa Cruz), or Gapdh (Santa Cruz).
The relationship is, however, not simple, since experiments with genetically marked myoblasts suggest that single myoblast clones can contribute to both fast and slow fibres, clones are not restricted to contribute to subsets of fibre types, and clones show no detectable preference for fusion to a particular fibre type (Hughes & Blau, 1992).
Mechanical forces are also thought to regulate growth in the case of "cell competition" in the Drosophila wing disc, in which slower growing clones of cells are outcompeted by faster growing neighbors, ultimately leading to apoptosis of the smaller clones, thereby ensuring uniform growth of the wing disc (Shraiman 2005).
Therefore, slow-growing clones will not be completely outcompeted when grown on a plate, and the phage produced by such clones will not be lost to the same extent as when grown in solution.
And he suggested he would like to move slower with cloning.
