Persistence of RNAi-Mediated Knockdown inDrosophilaComplicates Mosaic Analysis Yet Enables Highly Sensitive Lineage Tracing

Abstract The CreERT2/loxP system is widely used to induce conditional gene deletion in mice. One of the main advantages of the system is that Cre-mediated recombination can be controlled in time through Tamoxifen administration. This has allowed researchers to study the function of embryonic lethal genes at later developmental timepoints. In addition, CreERT2 mouse lines are commonly used in combination with reporter genes for lineage tracing and mosaic analysis. In order for these experiments to be reliable, it is crucial that the cell labeling approach only marks the desired cell population and their progeny, as unfaithful expression of reporter genes in other cell types or even unintended labeling of the correct cell population at an undesired time point could lead to wrong conclusions. Here we report that all CreERT2 mouse lines that we have studied exhibit a certain degree of Tamoxifen-independent, basal, Cre activity. Using Ai14 and Ai3, two commonly used fluorescent reporter genes, we show that those basal Cre activity levels are sufficient to label a significant amount of cells in a variety of tissues during embryogenesis, postnatal development and adulthood. This unintended labelling of cells imposes a serious problem for lineage tracing and mosaic analysis experiments. Importantly, however, we find that reporter constructs differ greatly in their susceptibility to basal CreERT2 activity. While Ai14 and Ai3 easily recombine under basal CreERT2 activity levels, mTmG and R26R-EYFP rarely become activated under these conditions and are therefore better suited for cell tracking experiments.

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A mosaic analysis system with Cre or Tomato expression in the mouse

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2014308117 ◽

2020 ◽

Vol 117 (45) ◽

pp. 28212-28220 ◽

Cited By ~ 1

Author(s):

Qun Wang ◽

Yen-Yu Lin ◽

Baojun Zhang ◽

Jianxuan Wu ◽

Sumedha Roy ◽

...

Keyword(s):

Somatic Mutations ◽

Reference Group ◽

Subsequent Development ◽

Lineage Tracing ◽

Clonal Hematopoiesis ◽

Age Related ◽

Mosaic Analysis ◽

Analysis System ◽

Mutant Cells

Somatic mutations are major genetic contributors to cancers and many other age-related diseases. Many disease-causing somatic mutations can initiate clonal growth prior to the appearance of any disease symptoms, yet experimental models that can be used to examine clonal abnormalities are limited. We describe a mosaic analysis system with Cre or Tomato (MASCOT) for tracking mutant cells and demonstrate its utility for modeling clonal hematopoiesis. MASCOT can be induced to constitutively express either Cre-GFP or Tomato for lineage tracing of a mutant and a reference group of cells simultaneously. We conducted mosaic analysis to assess functions of theId3and/orTet2gene in hematopoietic cell development and clonal hematopoiesis. Using Tomato-positive cells as a reference population, we demonstrated the high sensitivity of this system for detecting cell-intrinsic phenotypes during short-term or long-term tracking of hematopoietic cells. Long-term tracking ofTet2mutant orTet2/Id3double-mutant cells in our MASCOT model revealed a dynamic shift from myeloid expansion to lymphoid expansion and subsequent development of lymphoma. This work demonstrates the utility of the MASCOT method in mosaic analysis of single or combined mutations, making the system suitable for modeling somatic mutations identified in humans.

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The Q System: A Repressible Binary System for Transgene Expression, Lineage Tracing, and Mosaic Analysis

Cell ◽

10.1016/j.cell.2010.02.025 ◽

2010 ◽

Vol 141 (3) ◽

pp. 536-548 ◽

Cited By ~ 317

Author(s):

Christopher J. Potter ◽

Bosiljka Tasic ◽

Emilie V. Russler ◽

Liang Liang ◽

Liqun Luo

Keyword(s):

Binary System ◽

Transgene Expression ◽

Lineage Tracing ◽

System A ◽

Mosaic Analysis ◽

Q System

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Ovarian Dysfunction in Fetally Irradiated Beagles

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100080717 ◽

1977 ◽

Vol 35 ◽

pp. 658-659

Author(s):

T. M. Seed ◽

M. H. Sanderson ◽

D. L. Gutzeit ◽

T. E. Fritz ◽

D. V. Tolle ◽

...

Keyword(s):

Gamma Rays ◽

Low Dose ◽

Long Term Effects ◽

Ovarian Dysfunction ◽

Dose Rates ◽

Highly Sensitive ◽

Microscopic Evaluation ◽

Ovarian Pathology ◽

Normal Offspring

The developing mammalian fetus is thought to be highly sensitive to ionizing radiation. However, dose, dose-rate relationships are not well established, especially the long term effects of protracted, low-dose exposure. A previous report (1) has indicated that bred beagle bitches exposed to daily doses of 5 to 35 R 60Co gamma rays throughout gestation can produce viable, seemingly normal offspring. Puppies irradiated in utero are distinguishable from controls only by their smaller size, dental abnormalities, and, in adulthood, by their inability to bear young.We report here our preliminary microscopic evaluation of ovarian pathology in young pups continuously irradiated throughout gestation at daily (22 h/day) dose rates of either 0.4, 1.0, 2.5, or 5.0 R/day of gamma rays from an attenuated 60Co source. Pups from non-irradiated bitches served as controls. Experimental animals were evaluated clinically and hematologically (control + 5.0 R/day pups) at regular intervals.

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Fluorescence ratio imaging of dynamic intracellular ionic signals

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102298 ◽

1988 ◽

Vol 46 ◽

pp. 42-43

Author(s):

R. Y. Tsien ◽

A. Minta ◽

M. Poenie ◽

J.P.Y. Kao ◽

A. Harootunian

Keyword(s):

Binding Sites ◽

Living Cells ◽

Molecular Engineering ◽

Ph Indicators ◽

Highly Sensitive ◽

Fluorescence Ratio Imaging ◽

Ratio Imaging ◽

Technical Advances ◽

Indicator Dyes ◽

Fluorescein Dyes

Recent technical advances now enable the continuous imaging of important ionic signals inside individual living cells with micron spatial resolution and subsecond time resolution. This methodology relies on the molecular engineering of indicator dyes whose fluorescence is strong and highly sensitive to ions such as Ca2+, H+, or Na+, or Mg2+. The Ca2+ indicators, exemplified by fura-2 and indo-1, derive their high affinity (Kd near 200 nM) and selectivity for Ca2+ to a versatile tetracarboxylate binding site3 modeled on and isosteric with the well known chelator EGTA. The most commonly used pH indicators are fluorescein dyes (such as BCECF) modified to adjust their pKa's and improve their retention inside cells. Na+ indicators are crown ethers with cavity sizes chosen to select Na+ over K+: Mg2+ indicators use tricarboxylate binding sites truncated from those of the Ca2+ chelators, resulting in a more compact arrangement of carboxylates to suit the smaller ion.

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Various technic for the measurement of step thickness on crystal surfaces

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100109331 ◽

1978 ◽

Vol 36 (1) ◽

pp. 438-439

Author(s):

C. Boulesteix ◽

C. Colliex ◽

C. Mory ◽

B. Pardo ◽

D. Renard

Keyword(s):

Critical Thickness ◽

Symmetric Case ◽

Transmitted Intensity ◽

Crystal Surfaces ◽

Bright Field ◽

Excited Wave ◽

Highly Sensitive ◽

Contrast Mechanisms ◽

Step Thickness ◽

Thickness Variations

Contrast mechanisms, which are responsible of the various types of image formation, are generally thickness dependant. In the following, two imaging modes in the 100 kV CTEM are described : they are highly sensitive to thickness variations and can be used for quantitative estimations of step heights.Detailed calculations (1) of the bright-field intensity have been carried out in the 3 (or 2N+l)-beam symmetric case. They show that in given conditions, the two important symmetric Bloch waves interfere most strongly at a critical thickness for which they have equal emergent amplitudes (the more excited wave at the entrance surface is also the more absorbed). The transmitted intensity I for a Nd2O3 specimen has been calculated as a function of thickness t. The capacity of the method to detect a step and measure its height can be more clearly deduced from a plot of dl/Idt as shown in fig. 1.

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Estimation of the noise in TEM image recorded on “imaging plate”

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100128055 ◽

1987 ◽

Vol 45 ◽

pp. 748-749

Author(s):

T. Oikawa ◽

N. Mori ◽

T. Katoh ◽

Y. Harada ◽

J. Miyahara ◽

...

Keyword(s):

Low Dose ◽

Quantum Noise ◽

Laser Light ◽

Imaging Plate ◽

Total System ◽

Electron Dose ◽

X Ray ◽

Image Recording ◽

Recording Material ◽

Highly Sensitive

The “Imaging Plate”(IP) is a highly sensitive image recording plate for X-ray radiography. It has been ascertained that the IP has superior properties and high practicability as an image recording material in a TEM. The sensitivity, one of the properties, is about 3 orders higher than that of conventional photo film. The IP is expected to be applied to low dose techniques. In this paper, an estimation of the quantum noise on the TEM image which appears in case of low electron dose on the IP is reported.In this experiment, the JEM-2000FX TEM and an IP having the same size as photo film were used.Figure 1 shows the schematic diagram of the total system including the TEM used in this experiment. In the reader, He-Ne laser light is scanned across the IP, then blue light is emitted from the IP.

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High-sensitivity detection of gold labels by high-angle dark-field STEM imaging

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162028 ◽

1990 ◽

Vol 48 (3) ◽

pp. 892-893 ◽

Cited By ~ 1

Author(s):

Max T. Otten

Keyword(s):

High Sensitivity ◽

Dark Field ◽

Bright Field ◽

Backscattered Electron Imaging ◽

Backscattered Electrons ◽

Average Atomic Number ◽

Highly Sensitive ◽

Backscattered Electron ◽

Electron Imaging ◽

High Sensitivity Detection

Labelling of antibodies with small gold probes is a highly sensitive technique for detecting specific molecules in biological tissue. Larger gold probes are usually well visible in TEM or STEM Bright-Field images of unstained specimens. In stained specimens, however, the contrast of the stain is frequently the same as that of the gold labels, making it virtually impossible to identify the labels, especially when smaller gold labels are used to increase the sensitivity of the immunolabelling technique. TEM or STEM Dark-Field images fare no better (Figs. 1a and 2a), again because of the absence of a clear contrast difference between gold labels and stain.Potentially much more useful is backscattered-electron imaging, since this will show differences in average atomic number which are sufficiently large between the metallic gold and the stains normally used. However, for the thin specimens and at high accelerating voltages of the STEM, the yield of backscattered electrons is very small, resulting in a very weak signal. Consequently, the backscattered-electron signal is often too noisy for detecting small labels, even for large spot sizes.

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