4 ms·

Extended data figure 1h is testing the optimum pulse settings for the electroporation machine. There is a well-known trade-off between efficiency and viability

Extended data figure 1h is testing the optimum pulse settings for the electroporation machine. There is a well-known trade-off between efficiency and viability (too small a pulse and no DNA is transferred into cells, too strong a pulse and the cells are destroyed). The efficiency increase at lower viability cannot be attributed to pre-existing mutants, because they're assaying for the presence of a GFP transgene inserted by homology-directed repair, not indels in general.

nonbel 8y ago
>"The efficiency increase at lower viability cannot be attributed to pre-existing mutants, because they're assaying for the presence of a GFP transgene inserted by homology-directed repair, not indels in general." Yes, for the HDR case it is usually extremely unlikely (barring contamination) that the mutant already existed. If you reread my other post I specifically refer to the NHEJ case when discussing pre-existing mutants. However, a single T-cell could (T-cells can divide up to 12 times a day[1]) expand to as many as 2^48 = 2.81475e+14 daughter cells in the 4 days of that study (see panel A of the same figure). They started with only 1e6 cells in the culture. Therefore these results could be explained by editing a single cell that then proliferates to make up the 30% of the population (300k cells) they see. [1] http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0015423 http://journals.plos.org/plosone/article?id=10.1371/journal....
- azeotropic 8y ago
  Try reading the methods section of your T-cell paper. After electroporation, they cultured at constant cell density, which only required adding media every 2-3 days. So, on average, only one cell division happened before measurement, unless you want to claim GFP stimulates T-cells to hyperproliferate. My intuition is that cells with the transgene will actually be slower than their competitors, since they have to waste energy to make GFP. The PLoS One paper you've linked is about in-vivo hyperproliferation of T-cells in response to an ongoing infection, not about in-vitro primary culture in minimal media.
  - nonbel 8y ago
    >"So, on average, only one cell division happened before measurement, unless you want to claim GFP stimulates T-cells to hyperproliferate. My intuition is that cells with the transgene will actually be slower than their competitors, since they have to waste energy to make GFP." No, the claim is that double stranded breaks lead to cell death and senescence amongst the non-mutated cells thus selecting for the cells that lack the target sequence (because they were just "edited" or otherwise). We don't know how many cells survived the treatment, or how fast the different populations were dividing, or the rate at which these cells were dying. From extended fig 1a just see that 2 days after treatment there were ~500k total cells from an original ~1 million and ~100k of those expressing GFP. Then four days after treatment there were ~1 million total cells with ~ 300k of them expressing GFP. Are T-cells capable of dividing quick enough so a single cell can generate 100k in 2 days and 300k in 4 days? The literature says yes. Maybe there was an initial thousand edited cells that divided on average ~3 times each day for two days and then once the last two days as the surviving non-GFP+ cells recovered. Is that what happened? I don't know. They didnt report the details of what happened immediately after the treatment. >"The PLoS One paper you've linked is about in-vivo hyperproliferation of T-cells in response to an ongoing infection, not about in-vitro primary culture in minimal media." Yes, it only demonstrates how fast they could be dividing, setting an upper bound. We don't know under the conditions in this paper.

nonbel 8y ago

>"The efficiency increase at lower viability cannot be attributed to pre-existing mutants, because they're assaying for the presence of a GFP transgene inserted by homology-directed repair, not indels in general." Yes, for the HDR case it is usually extremely unlikely (barring contamination) that the mutant already existed. If you reread my other post I specifically refer to the NHEJ case when discussing pre-existing mutants. However, a single T-cell could (T-cells can divide up to 12 times a day[1]) expand to as many as 2^48 = 2.81475e+14 daughter cells in the 4 days of that study (see panel A of the same figure). They started with only 1e6 cells in the culture. Therefore these results could be explained by editing a single cell that then proliferates to make up the 30% of the population (300k cells) they see. [1] http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0015423 http://journals.plos.org/plosone/article?id=10.1371/journal....

azeotropic 8y ago
Try reading the methods section of your T-cell paper. After electroporation, they cultured at constant cell density, which only required adding media every 2-3 days. So, on average, only one cell division happened before measurement, unless you want to claim GFP stimulates T-cells to hyperproliferate. My intuition is that cells with the transgene will actually be slower than their competitors, since they have to waste energy to make GFP. The PLoS One paper you've linked is about in-vivo hyperproliferation of T-cells in response to an ongoing infection, not about in-vitro primary culture in minimal media.
- nonbel 8y ago
  >"So, on average, only one cell division happened before measurement, unless you want to claim GFP stimulates T-cells to hyperproliferate. My intuition is that cells with the transgene will actually be slower than their competitors, since they have to waste energy to make GFP." No, the claim is that double stranded breaks lead to cell death and senescence amongst the non-mutated cells thus selecting for the cells that lack the target sequence (because they were just "edited" or otherwise). We don't know how many cells survived the treatment, or how fast the different populations were dividing, or the rate at which these cells were dying. From extended fig 1a just see that 2 days after treatment there were ~500k total cells from an original ~1 million and ~100k of those expressing GFP. Then four days after treatment there were ~1 million total cells with ~ 300k of them expressing GFP. Are T-cells capable of dividing quick enough so a single cell can generate 100k in 2 days and 300k in 4 days? The literature says yes. Maybe there was an initial thousand edited cells that divided on average ~3 times each day for two days and then once the last two days as the surviving non-GFP+ cells recovered. Is that what happened? I don't know. They didnt report the details of what happened immediately after the treatment. >"The PLoS One paper you've linked is about in-vivo hyperproliferation of T-cells in response to an ongoing infection, not about in-vitro primary culture in minimal media." Yes, it only demonstrates how fast they could be dividing, setting an upper bound. We don't know under the conditions in this paper.