Skip to main content
NCBI home page
Journal of Anatomy logoLink to Journal of Anatomy
. 2005 Jan;206(1):79–92. doi: 10.1111/j.0021-8782.2005.00370.x

Evaluation of the mouse TgTP6.3 tauGFP transgene as a lineage marker in chimeras

Gillian E MacKay 1,*, Margaret A Keighren 1,†, Linda Wilson 2, Thomas Pratt 2, Jean H Flockhart 1, John O Mason 2, David J Price 2, John D West 1
PMCID: PMC1571452  PMID: 15679873

Abstract

The mouse TgTP6.3 transgene, encoding a tauGFP fusion protein, is becoming widely used but has yet to be fully characterized and evaluated as suitable lineage marker. The aim of the present study was to investigate the phenotype of TgTP6.3+/+ homozygotes and TgTP6.3+/– hemizygotes, characterize the expression of the TgTP6.3 transgene in different tissues and critically evaluate its use as a lineage marker. TgTP6.3+/+ homozygotes died between embryonic day 14.5 and weaning, whereas TgTP6.3+/– hemizygotes were mostly viable and fertile but smaller than non-transgenic siblings. TgTP6.3 expression began in the late two-cell stage, persisted in most fetal and adult tissues and was uniformly expressed in many (but not all) tissues. TgTP6.3+/– cells were readily identified in many chimeric tissues and their contribution appeared to be quantitatively and spatially normal. Overall, tauGFP expression in hemizygous TgTP6.3+/– cells fulfils the main criteria of a good lineage marker for many tissues. It provides a useful lineage marker, which should be particularly suitable for axons, blood vessels and pre-implantation embryos.

Keywords: chimera, green fluorescent protein, lineage marker, chimaera, transgene

Introduction

Green fluorescent protein (GFP) has great potential as a lineage marker and several investigators have produced transgenic mice that express GFP or related fluorescent proteins in a wide range of cell types (Hadjantonakis et al. 2003). Early studies with GFP transgenics were suboptimal because the original wild-type GFP was neither stable nor highly fluorescent at mouse body temperature (Ikawa et al. 1998).Zernicka-Goetz et al. (1997) used mutant GFP (MmGFP) driven by a PGK promoter to produce mouse chimeras from GFP-positive embryonic stem cells, but the transgene was not transmitted through the germ line so no green mice were produced. The first live GFP transgenic mouse was produced using the ubiquitous CAG promoter [cytomegalovirus (CMV) enhancer, chicken β-actin promoter fragment and rabbit β-globin exons] but GFP expression was not ubiquitous and brain and blood vessels were usually negative (Ikawa et al. 1995). When mutant EGFP (enhanced GFP) was used with the same CAG promoter, however, expression was virtually ubiquitous, except for hair and red blood cells (Okabe et al. 1997; Ikawa et al. 1998).Hadjantonakis et al. (1998) produced several transgenic lines expressing the EGFP transgene including strain Tg(GFPU)5Nagy, which showed ubiquitous expression but with levels that varied among organs (fluorescence was obscured by pigmented hair, masked by haemoglobin and appeared weak in blood vessels, spleen, kidney and liver).

Pratt et al. (2000) produced a line of mice (TP6.3) carrying the TgTP6.3 transgene that expressed GFP fused to the microtubule-associated protein tau (tauGFP), driven by the CAG promoter. TgTP6.3+/– hemizygotes showed ubiquitous tauGFP expression at the four-cell stage and expression was widespread and readily detectable in fresh and fixed tissue samples of several prenatal and postnatal stages. Expression of GFP as a tauGFP fusion protein provided an excellent marker for axons but the widespread GFP expression also made TgTP6.3 a good candidate for use as a lineage marker in many other tissues. Moreover, preservation of tauGFP after fixation appeared to be better than for soluble forms of GFP, making it more suitable for analysis of tissue sections. TP6.3 mice are becoming more widely used (e.g.Ying et al. 2003; Court et al. 2004) but have yet to be fully characterized. The original report of the TgTP6.3 transgene (Pratt et al. 2000) focused on embryonic stem (ES) cells, embryonic brain expression and axonal labelling but did not evaluate it as a suitable lineage marker for other tissues.

As discussed elsewhere (McLaren, 1976; West, 1984), good lineage markers should be (1) retained within cells, (2) not transferred between cells, (3) widely expressed, (4) easy to detect in fresh and fixed material, (5) stable and expressed uniformly throughout development, and (6) developmentally neutral. The earlier investigations of Pratt et al. (2000) showed that expression of TgTP6.3 in hemizygous mice fulfilled the first four of these criteria. However, it was unclear from this first study whether TgTP6.3 expression was uniform throughout development or was developmentally neutral.

The aim of the present study was to characterize the TP6.3 transgenic line in more detail and determine whether TgTP6.3 expression is suitable for lineage analysis experiments in a range of tissues. The viability and growth characteristics of homozygous TgTP6.3+/+ and hemizygous TgTP6.3+/– mice were investigated, and tauGFP expression was examined in different tissues. Finally, the contribution of TgTP6.3+/– cells to tissues of chimeric mice was evaluated to determine whether TgTP6.3 is developmentally neutral.

Materials and methods

Mice

The tauGFP, TgTP6.3 transgene (Pratt et al. 2000) was bred onto different genetic backgrounds. Hemizygous TgTP6.3+/– mice that were pigmented and homozygous Gpi1b/b were selected and maintained by crossing to non-transgenic (C57BL/Hsd × CBA/Ca)F1 hybrid mice (TgTP6.3–/–) purchased from the Anne Walker animal facility, University of Edinburgh. TgTP6.3+/– mice were also crossed to (BALB/c × A/J)F1 mice and made albino and homozygous for Gpi1a. Inbred BALB/c and A/J mice were purchased from Harlan Olac and (BALB/c × A/J)F1 hybrid mice were bred from these stocks. In genetic crosses the female parental genotypes are shown first. The presence of the transgene is denoted by a plus sign and because they are addition transgenics the term ‘hemizygote’ is used rather than ‘heterozygote’. For example, TgTP6.3+/– × TgTP6.3–/– indicates a genetic cross between a hemizygous TgTP6.3+/– transgenic female and a wild-type male. Mice were bred and maintained under conventional conditions at the University of Edinburgh, with a 14-h light (05:00–19:00 h) 10-h dark (19:00–05:00 h) cycle.

Adult mice carrying the tauGFP transgene were identified by fluorescence microscopy of ear tissue obtained as a by-product of ear clipping for identification purposes. Fetuses were examined with a Leica MZ FLIII fluorescence stereomicroscope and some newborn pups were identified as described by Tyas et al. (2003).

Chimera production

Chimeras were produced by aggregating pairs of pre-implantation embryos produced from superovulated females using the method developed by Tarkowski (1961), as described elsewhere (West & Flockhart, 1994). Two series of chimeras were produced for analysis of the distribution of TgTP6.3+/– cells by confocal microscopy. Another two series were produced to test whether the presence of hemizygous TgTP6.3+/– cells affected the composition of mid-gestation (E12.5) chimeric conceptuses.

Chimeric blastocysts for confocal analysis were produced by aggregating (C57BL × CBA/Ca)F1 × TgTP6.3+/– eight-cell embryos (some were TgTP6.3+/– and tauGFP-positive; others were TgTP6.3–/– and tauGFP-negative) with tauGFP-negative (C57BL × CBA/Ca)F2 eight-cell embryos. For the production of fetal and adult chimeras for analysis by confocal microscopy, superovulated, albino (BALB/c × A/J)F1 females were crossed to albino, Gpi1a/a, TgTP6.3+/– males and eight-cell embryos recovered at embryonic day 2.5 (E2.5) in M2 handling medium (Quinn et al. 1982). Eight-cell embryos were aggregated with eight-cell embryos from crosses between superovulated (C57BL × CBA/Ca)F1 females and homozygous TgR(ROSA26)26Sor+/+ males. After overnight culture, the chimeric aggregates were transferred to the uteri of pseudopregnant females. E12.5 chimeric conceptuses, for glucose phosphate isomerase (GPI) analysis of the composition of fetuses and extra-embryonic tissues, were produced as described above but they differed in strain combination. (C57BL/Hsd × CBA/Ca)F1 females were crossed to pigmented, Gpi1b/b, hemizygous TgTP6.3+/– males and GFP-positive, eight-cell embryos were distinguished from their non-transgenic littermates by brief examination with a Leica TCS NT confocal microscope. These were aggregated either to eight-cell stage inbred BALB/c embryos or to (BALB/c × A/J)F2 embryos (both stocks were albino and Gpi1a/a). Chimeric aggregates were transferred to homozygous Gpi1c/c pseudopregnant females.

Analysis of GFP expression

Live pre-implantation stage embryos were viewed with an inverted confocal microscope in drops of M16 or KSOM culture medium or in M2 or KSOM-H handling medium under silicon oil (BDH) in glass-bottomed Willco microwell dishes (Intracel Ltd, HBSt 3522). In some cases, embryos were cultured for up to 24 h in a controlled environment chamber on the stage of the confocal microscope.

For fetal and adult tissues, we compared different types of specimen preparation and fluorescence microscopy. Vibratome sections proved to be superior to either frozen sections or standard wax-embedded sections. Standard fluorescence microscopy enabled the tauGFP fluorescence to be detected but was not suitable for resolving individual cells in thick sections or whole mounts because the fluorescence from several cell layers was superimposed. The optimum method for adult and fetal tissues was confocal microscopy of 100- to 200-µm-thick vibratome sections. Various fetal and adult tissues from TgTP6.3+/– hemizygotes and chimeras were dissected, fixed overnight in 4% paraformaldehyde, washed in two changes of PBS and embedded in 4% agarose. Sections 200 µm thick were cut with a vibratome, washed in PBS, immersed in a 1 : 1 solution of PBS and glycerol at 4 °C until saturated, and mounted in 1 : 1 PBS–glycerol containing 1% Vectashield antifade reagent (H-1000, Vector Laboratories Inc., USA). The coverslips were sealed with Aquamount. Some tissue sections were counterstained in propidium iodide (PI) solution (0.1% PI, 0.05% RNase, 0.1% Triton X-100 in PBS) for 30 min, prior to mounting, to counterstain the nuclei red. In some cases RNase was omitted and PI also stained the cytoplasm. Bright-field images were collected in the transmitted channel. GFP was detected in the FITC (green) channel.

Analysis of composition of chimeras by GPI electrophoresis

Recipient females were killed at 12.5 days of gestation and the E12.5 conceptuses were dissected to provide five samples: fetus, amnion, yolk sac mesoderm, yolk sac endoderm and placenta (West & Flockhart, 1994). All tissues were stored at −20 °C in 50% glycerol in water, 200 µL each for fetus and placenta in 1.5-mL Eppendorf microfuge tubes, 20 µL each for the other tissues in 96-well plates. Samples were lysed by three cycles of freezing–thawing with mechanical disruption. Each chimera was a mixture of homozygous Gpi1a/a and Gpi1b/b cells, and the recipient females were homozygous Gpi1c/c. The proportions of the two cell populations in the chimeric tissues were estimated from the proportions of GPI1-A and GPI1-B allozymes, after electrophoresis, staining for GPI1 activity and densitometry with a Helena Process-24 gel scanner (West & Flockhart, 1994). Any maternal contamination appeared as a GPI1-C band, which was excluded from the calculations. The percentage of eye pigment was estimated subjectively for fetal chimeras.

Results

Genetic linkage

TgTP6.3 has not yet been mapped to a specific chromosome but breeding records prove that it is not X-linked because hemizygous males transmitted the transgene to both male and female progeny of TgTP6.3–/– female × TgTP6.3+/– male crosses.

Growth and viability of hemizygotes

In the first experiment, offspring from TgTP6.3+/– hemizygous males and non-transgenic (C57BL × CBA/Ca)F1 females were sexed, tested for tauGFP expression and weighed at 3 weeks. All those surviving to 6 weeks and some of those surviving to 12 weeks were re-weighed. Both male and female TgTP6.3+/– hemizygous mice were significantly lighter than their non-transgenic littermates at each age (Fig. 1A,B). The small reduction in numbers weighed between 3 and 6 weeks reflects death of a few mice during this period. For males, the proportion of GFP-positive mice was 55/132 and 49/125 at 3 and 6 weeks, respectively. This was not significantly less than the 50% expected at 3 weeks (χ2 = 3.67) but was significantly less than 50% by 6 weeks (χ2 = 5.83; P < 0.05). For females, the proportion of GFP-positive mice was not less than 50% at either 3 weeks (66/122) or 6 weeks (57/111). Although some of the smaller GFP-positive mice died soon after weaning, the survivors developed normally.

Fig. 1.

Fig. 1

Open in a new tab

Comparison of weights of hemizygous GFP-positive and non-transgenic littermates from crosses between hemizygous males and non-transgenic (C57BL × CBA/Ca)F1 females (see text for explanation of the two experiments in A and B).(A)Postnatal weights of hemizygous TgTP6.3+/– and non-transgenic females. (B)Postnatal weights of hemizygous TgTP6.3+/– and non-transgenic males. (C)Weights of hemizygous TgTP6.3+/– and non-transgenic fetuses. The numbers of mice are shown close to the error bars in C and for experiment 1 in A and B. Statistical significance by unpaired t-tests are shown as P values in C and by asterisks in A and B (*P < 0.05; **P < 0.01; ***P < 0.0001).

This cross was repeated, in a second experiment, to allow weight changes to be monitored on a weekly basis. Mice were weighed on the day after birth (postnatal day 1, P1), P5 and then every 7 days until P89 (Fig. 1A,B). Female TgTP6.3+/– hemizygous mice were significantly lighter than non-transgenic female littermates from P5, with the exception of P19. Hemizygous males were significantly lighter than non-transgenic male littermates from P1.

In a third experiment, fetuses were classified for GFP expression and weighed but males and females were not distinguished. The results showed that hemizyous TgTP6.3+/– fetuses were significantly lighter than non-transgenic littermates at E14.5 and E17.5 but not at E12.5 (Fig. 1C).

These comparisons show that TgTP6.3+/– hemizygotes were significantly smaller than their non-transgenic littermates from late fetal stages to 12 weeks after birth. Some TgTP6.3+/– mice (mostly males) died shortly after weaning. This is attributed to small size at 3 weeks and survival appears to be improved by delaying weaning until after 3 weeks.

Lethality of homozygotes

In the absence of a simple molecular test, we used a genetic approach to test whether TgTP6.3+/+ homozygotes survived to reproductive age. Seventeen GFP-positive offspring of TgTP6.3+/– × TgTP6.3+/– intercrosses produced litters on test-crossing to non-transgenic mice. All 17 GFP-positive mice produced some GFP-negative offspring, proving that they were not homozygotes. If homozygotes and hemizygotes were equally viable and fertile, 33.3% of the GFP-positive mice would be expected to be homozygous. The probability that all 17 offspring tested were, by chance, hemizygotes is only (2/3)17 = 0.001. This implies that homozygous TgTP6.3+/+ mice either died before test breeding or were infertile and failed to produce offspring. Another two GFP-positive mice failed to reproduce after being paired in the test-cross for at least 2 months. This proportion (2/19) is also significantly less than the predicted 33.3% homozygotes (χ2 = 4.45; P < 0.05) so the results cannot adequately be explained by assuming that all homozygous TgTP6.3+/+ mice survive but are infertile. This suggests that TgTP6.3+/+ homozygotes died before reaching reproductive age.

Viability of fetal homozygotes

Conceptuses were examined at E14.5 to determine if homozygous fetuses were viable. No obvious differences in intensity of GFP fluorescence were noted among fetuses from experimental crosses (TgTP6.3+/– × TgTP6.3+/–) that should have produced both homozygous and hemizygous GFP-positive progeny. The frequency of GFP-positive E14.5 fetuses and the frequency of dead conceptuses were compared in these experimental crosses and control (TgTP6.3+/– female × TgTP6.3–/– non-transgenic male) crosses (Table 1). The percentage of death attributable to production of homozygotes in the experimental cross was calculated by correcting for sporadic death in the control cross, using the formula shown in Table 1(adapted from Lyon, 1970).

Table 1.

Analysis of viability of E14.5 TgTP6.3+/+ homozygous fetuses in the experimental TgTP6.3+/– × TgTP6.3+/– crosses.

Classification of conceptuses at E14.5 Control cross TgTP6.3+/– × TgTP6.3−/− Experimental cross TgTP6.3+/– × TgTP6.3+/−
Total conceptuses 201 215
Total normal fetuses 181 175
GFP-positive 105a(58.0%) 132b(75.4%)
GFP-negative  76 (42.0%)  43 (24.6%)
Moles (early deaths)  14 (7.0%)  33 (15.3%)
Dead fetuses   6 (3.0%)   7 (3.3%)
Death attributable to cross* NA   9.6%
Open in a new tab

NA, not applicable.

a,b

Proportion of GFP-positive progeny differs significantly by χ2 test from: a50% expected in control cross (P < 0.05), and b67% expected in experimental cross if all homozygotes die but all hemizygotes survive (P < 0.05).

*
The percentage of death attributable to production of GFP homozygotes in the experimental crosses was calculated by correcting for sporadic death in the control crosses as follows:
[1-(normal fetuses in experimental crosstotal conceptuses in experimental cross/normal fetuses in control crosstotal conceptuses in control cross)]×100

Table 1 shows that the percentage of GFP-positive E14.5 fetuses in the control cross was not less than the expected 50% (58.0%) and the total of the early and late deaths was only 10.0%, implying that they survived as well as non-transgenic siblings. For the experimental cross the proportions of GFP-positive fetuses was 75.4%. This is close to the 75% expected if the viability was similar for all genotypes and significantly higher than the 66.7% predicted if all homozygotes died by this stage. The total frequency of dead conceptuses in the experimental cross was significantly higher than in the control cross (40/215 vs. 20/201; χ2 = 5.62; P = 0.018). The lethality attributable to the difference between the experimental and control crosses was calculated as 9.6%, which is below the 25% expected if all homozygotes died. The proportions of GFP-positive fetuses and the frequencies of dead conceptuses indicate that most TgTP6.3+/+ homozygotes survived to E14.5 but the results do not exclude the possibility of slightly reduced viability of homozygotes.

Perinatal lethality of homozygotes

Table 2 shows results obtained retrospectively from records of pups born and weaned in our mouse colonies. The percentage of mice from the TgTP6.3–/– female × TgTP6.3+/– male control crosses that survived to weaning and expressed GFP (46.2%) was not significantly different from the predicted 50%. The percentage of mice from the TgTP6.3+/– × TgTP6.3+/– experimental crosses that survived to weaning and expressed GFP (54.4%) was significantly lower than both 75% and 66.7%. Significantly more pups died before weaning in the experimental cross than in the control cross (18/153 vs. 3/135; χ2 = 8.30; P = 0.004) and it was calculated that 9.8% of preweaning deaths in the experimental cross were attributable to differences from the control cross (probably the presence of homozygotes).

Table 2.

Retrospective analysis of viability of TgTP6.3+/+ homozygotes from breeding records of control TgTP6.3−/– female × TgTP6.3+/– male and experimental TgTP6.3+/– × TgTP6.3+/– crosses

Classification of progeny* Control cross TgTP6.3−/– × TgTP6.3+/− Experimental cross TgTP6.3+/– × TgTP6.3+/−
Total born 135 153
GFP-positive  54 (46.2%)  68ab(54.4%)
GFP-negative  63 (53.8%)  57 (45.6%)
GFP not classified  13   9
Dead before weaning   3 (2.2%)  18 (11.8%)
Dead after weaning   2 (1.5%)   1 (0.7%)
Pre-weaning death attributable to cross† NA   9.8%
Open in a new tab

NA, not applicable.

a,b

Proportion of GFP-positive progeny differs significantly by χ2 test from: a75% expected in experimental cross if all are viable (P < 0.001), and b67% expected in experimental cross if all homozygotes die but all hemizygotes survive (P < 0.005).

*

GFP was classified approximately 3 weeks after birth.

†
The percentage of death attributable to production of GFP homozygotes in the experimental crosses was calculated by correcting for sporadic death in the control crosses as follows:
[1-(viable mice in experimental crosstotal born in experimental cross/viable mice in control crosstotal born in control cross)]×100

Overall, the comparisons between control and experimental crosses at E14.5 and after birth indicated that most TgTP6.3+/+ homozygotes survived to E14.5 but died before weaning. Although the estimated incidence of homozygous death between birth and weaning did not reach the 25% expected if all homozygotes died during this period, the extent of this lethality may have been underestimated if some pups died and were eaten before births were recorded. It is possible that lethality occurs throughout a broad perinatal period (from late fetal to early postnatal stages).

GFP expression in hemizygous and chimeric pre-implantation embryos

Pratt et al. (2000) reported that TgTP6.3+/– hemizygotes that inherited the transgene paternally showed ubiquitous tauGFP expression from the four-cell stage. We confirmed this and also found paternal expression in both blastomeres in some late two-cell stage embryos (Fig. 2A–D). As the TgTP6.3 transgene is expressed at these early stages, TgTP6.3 was evaluated as a marker in pre-implantation stage TgTP6.3+/–↔TgTP6.3–/– chimeras. TgTP6.3 provided an excellent marker for pre-implantation stage chimeras (Fig. 2E,F) and the tauGFP-positive cells can be visualized very clearly with the confocal microscope. As shown in other studies (Garner & McLaren, 1974; Dvorak et al. 1995; Everett et al. 2000) there was very little mixing between the two aggregated embryos in chimeric blastocysts.

Fig. 2.

Fig. 2

Open in a new tab

Early embryonic stages. (A–D) Expression of tauGFP in a single batch of pre-implantation stage embryos, from a cross between non-transgenic (C57BL × CBA/Ca)F1 females and hemizygous TgTP6.3+/– males, progressing from the two-cell stage (A,B) to the four-cell stage (C,D). Embryos were viewed under conditions to show (A,C) GFP fluorescence, and (B,D) transmitted light image. The GFP-positive, TgTP6.3+/– embryos are clearly distinguishable from GFP-negative, TgTP6.3–/– embryos at the four-cell stage and comparison between A and C shows that GFP expression is faintly visible in some of the two-cell stage embryos (e.g. arrows). (E,F) Pre-implantation stage TgTP6.3+/–↔TgTP6.3–/– chimeras. (E) GFP fluorescence combined with transmitted light image of blastocyst stage chimeras (some chimeras do not have a TgTP6.3+/– cell population). (F) GFP fluorescence produced by chimeric blastocyst (GFP-negative cells are invisible). (G–J) E6.5 (G) and E7.5 (H–J) egg cylinder stage, post-implantation TgTP6.3+/– embryos viewed with different filters to allow visualization of both GFP and propidium iodide (G,H) or GFP alone (I,J). GFP expression is strongest in the epiblast-derivatives and trophoblast giant cells of egg cylinder stage embryos. Labels: ex, extra-embryonic ectoderm; m, mesoderm, tgc, trophoblast giant cells. Scale bars in A–E and G–I = 100 µm; F and J = 50 µm.

GFP expression in hemizygous early post-implantation embryos

Vibratome sections of hemizygous TgTP6.3+/– early post-implantation embryos (E6.5 and E7.5 egg cylinder stages) showed ubiquitous GFP expression but the signal intensity was not uniform and was stronger in the epiblast derivatives than in the tissues derived from the hypoblast or trophectoderm (Fig. 2G–J). Nevertheless, GFP signal was strong in invading trophoblast giant cells at E6.5 and E7.5 (Fig. 2G–I) and was strong in E10.5 placental trophoblast (see Fig. 4D).

Fig. 4.

Fig. 4

Open in a new tab

Tissues from fetal (D) and adult (A–C and E–L) TgTP6.3+/– hemizygotes: (A) brain; (B) liver; (C) kidney; (D) E10.5 placenta; (E) brain (showing strong GFP fluorescence in blood vessels); (F) adrenal gland (showing several radial stripes of cells with little or no GFP expression in the cortex); (G–I) testis; (J–L) ovary. GFP expression differs markedly between different cell types in the ovary and testis (see text). Viewed with different filters to allow visualization of GFP alone (A–G,J), propidium iodide alone (H,K) or both GFP and propidium iodide (I,L). Scale bars: A = 1000 µm; B–L = 200 µm.

GFP expression in hemizygous and chimeric fetal tissues

A series of TgTP6.3+/–↔TgTP6.3–/– aggregation chimeras was produced and analysed by confocal microscopy at fetal and adult stages to determine whether the TgTP6.3+/– hemizygous cells could be clearly identified and whether they were distributed normally in different tissues. Confocal images of an E14.5 non-chimeric TgTP6.3+/– fetal heart (Fig. 3A–C) showed it was entirely green, although some areas fluoresced more brightly than others. In contrast, comparable images of an E14.5 fetal chimeric heart revealed distinct patches of GFP-positive and GFP-negative cells (Fig. 3D–F). GFP signal was particularly strong in the non-chimeric E13.5 fetal lens (Fig. 3G). Figure 3(H,I) shows the distribution of GFP-positive cells in E12.5 chimeric fetal eyes. Patches of GFP-positive and negative cells were seen in the lens epithelium, lens fibres and neural retina.

Fig. 3.

Fig. 3

Open in a new tab

Fetal tissues from TgTP6.3+/– hemizygotes and chimeras: (A–C) E14.5 hemizygous fetal heart, (D–F) E14.5 chimeric fetal heart, (G) E13.5 hemizygous fetal eye, (H,I) two different E12.5 chimeric fetal eyes. Viewed with different filters for visualization of GFP alone (A,D,G,H), propidium iodide alone (B,E) or both GFP and propidium iodide (C,F,I). Patches of GFP-positive and GFP-negative cells are clearly visible in the chimeric tissues. Scale bars = 100 µm.

GFP expression in hemizygous adult tissues

GFP was expressed in all non-chimeric hemizygous adult organs examined, including the brain, liver, kidney, adrenal gland, testis, ovary, lungs, heart and eye; examples are illustrated in Fig. 4. Signal was fairly uniform in the non-chimeric liver (Fig. 4B), lungs, heart and eye. However, the signal was not uniform in all organs. For example, in the kidney GFP signal was strongest in the glomeruli (Fig. 4C). In the testis (Fig. 4G–I), interstitial cells fluoresced strongly whereas germ cells and other cells within the seminiferous tubules showed little or no GFP signal. In the ovary (Fig. 4J–L), corpora lutea showed strong fluorescence, interstitial cells fluoresced more weakly and fluorescence was extremely weak in the follicles and oocytes. Unlike many other GFP markers, tauGFP proved particularly good for highlighting blood vessels (Fig. 4E).

GFP was sometimes expressed evenly throughout the adrenal cortex but expression was often mosaic, with stripes of non-expressing cells spanning the cortex (Fig. 4F). These stripes presumably reflect clones of cells that have down-regulated the transgene. TgTP6.3+/– is therefore not suitable as a lineage marker in TgTP6.3+/–↔TgTP6.3–/– chimeric adrenal tissue as some GFP-negative stripes will be from non-transgenic TgTP6.3–/– cells and some will be from hemizygous TgTP6.3+/– cells.

GFP expression in postnatal chimeric tissues

Figure 5 shows the distribution of GFP-positive cells in tissues from postnatal chimeras. TgTP6.3+/– provided an excellent marker for chimeric liver (Fig. 5A) and heart (Fig. 5B) as GFP-positive cells and GFP-negative cells could be clearly distinguished. GFP-positive stripes from transgenic TgTP6.3+/– cells and GFP-negative stripes from non-transgenic TgTP6.3–/– cells spanned the chimeric neural retina (Fig. 5C) as previously reported for other markers (Reese et al. 1999; Collinson et al. 2001). However, we have not investigated whether GFP is expressed in all cell types of the neural retina. The hemizygous TgTP6.3+/– and non-transgenic TgTP6.3–/– cells were not readily distinguished in chimeric kidney, ovarian follicles and testis, because GFP signal was not uniform in non-chimeric kidneys and GFP signal was weak or absent in the ovarian follicles and testicular tubules (Fig. 4). Hemizygous TgTP6.3+/– cells made a good contribution to postnatal chimeras. Although this was not studied systematically, the contribution of GFP-positive cells was estimated in the liver, tail and blood of two chimeras by GPI electrophoresis. The GFP-positive contribution in these samples ranged from 28% to 69% and the mean for each chimera was 39% and 60%, respectively. The absence of any consistent deficiency or excess of TgTP6.3+/– cells is compatible with developmental neutrality.

Fig. 5.

Fig. 5

Open in a new tab

Tissues from adult TgTP6.3+/–↔TgTP6.3–/– chimeras: (A) liver, (B) heart, (C) neural retina. Patches of GFP-positive cells are clearly visible in each tissue and in the neural retina they form stripes that span the thickness of the entire neural retina. Viewed with different filters to allow visualization of both GFP and propidium iodide. Scale bars = 200 µm.

Quantitative composition of TgTP6.3+/–↔TgTP6.3–/– chimeric conceptuses

The composition of aggregation chimeras is affected by the strain combination and some strain combinations are consistently unbalanced so that one strain predominates in most individual chimeras (Mullen & Whitten, 1971). Two series of E12.5 TgTP6.3+/–↔TgTP6.3–/– chimeras were produced to test whether incorporation of TgTP6.3+/– hemizygous cells into chimeras perturbed the normal strain-dependent composition of chimeric conceptuses. TgTP6.3–/– and TgTP6.3+/– embryos were produced from (C57BL × CBA/Ca)F1 × TgTP6.3+/– matings but only TgTP6.3+/– hemizygotes (identified by fluorescence microscopy) were used for the aggregations. In one series TgTP6.3+/– embryos were aggregated to non-transgenic TgTP6.3–/– embryos from the inbred BALB/c strain to produce BALB/c↔(C57BL × CBA/Ca)F1 × TgTP6.3+/– chimeras. Previous studies have shown that BALB/c strain embryos tend to make a poor contribution to chimeras (Mullen & Whitten, 1971; West & Flockhart, 1994; Tang & West, 2001) so this series of chimeras was expected to be unbalanced, with hemizygous TgTP6.3+/– cells predominating, unless the expression of tauGFP adversely affected development. The other series was made in a similar way except that (BALB/c × A/J)F2 non-transgenic embryos were used instead of inbred BALB/c embryos. This (BALB/c × A/J)F2↔(C57BL × CBA/Ca)F1 × TgTP6.3+/– series was expected to be genotypically balanced if TgTP6.3+/– cells were developmentally neutral. Each series of chimeras was genetically albino, Gpi1a/a, TgTP6.3–/–↔pigmented, Gpi1b/b, TgTP6.3+/– and the contribution of TgTP6.3+/– (Gpi1b/b) cells in the E12.5 fetus and extra-embryonic tissues was estimated by electrophoresis of GPI allozymes.

The mean percentage GPI1-A was analysed in chimeric and non-chimeric conceptuses produced in these two new series and compared with two previously published pairs of balanced and unbalanced strain combinations. For each pair, the unbalanced strain combination involved inbred BALB/c embryos (homozygous Gpi1a/a), which tend to contribute poorly to chimeras. Unbalanced strain combinations tend to produce a higher proportion of non-chimeric conceptuses than balanced strain combinations (West & Flockhart, 1994). This was apparent in the new series of BALB/c↔(C57BL × CBA/Ca)F1 × TgTP6.3+/– chimeras (one GPI1-A, 20 chimeric and seven GPI1-B conceptuses). Most of the non-chimeric conceptuses were GPI1-B (homozygous Gpi1b/b, non-BALB/c genotype). In contrast, this was not seen in the new series of (BALB/c × A/J)F2↔ × (C57BL × CBA/Ca)F1 × TgTP6.3+/– chimeras (two GPI1-A, 20 chimeras and two GPI1-B).

The composition of the fetuses and extra-embryonic tissues in the conceptuses that were chimeric is shown in Fig. 6. In each experiment the unbalanced strain combination had a lower contribution of GPI1-A cells in all tissues. These results show that the hemizygous TgTP6.3+/– embryos, from (C57BL × CBA/Ca)F1 × TgTP6.3+/– crosses, contributed normally in chimeras. They formed a balanced series of chimeras in combination with (BALB/c × A/J)F2 embryos and unbalanced chimeras in combination with inbred BALB/c embryos. In this respect the hemizygous TgTP6.3+/– cells behaved similarly to (C57BL × CBA/Ca)F2 cells (West & Flockhart, 1994), implying that the TgTP6.3+/– genotype is developmentally neutral.

Fig. 6.

Fig. 6

Open in a new tab

Comparison of the composition (mean percentage GPI1-A) of three pairs of E12.5 balanced and unbalanced chimera series. In each case the balanced series is shaded black and the unbalanced series is shaded grey. (A) Balanced series (C57BL-Gpi1a, c × BALB/c)F2↔(C57BL × CBA/Ca)F2 and unbalanced series BALB/c↔(C57BL × CBA/Ca)F2 (West & Flockhart, 1994). (B) Balanced series (BALB/c × A/J)F2↔(C57BL × CBA/Ca)F1 × TGB and unbalanced series BALB/c↔(C57BL × CBA/Ca)F1 × TGB (Tang & West, 2001). (C) New balanced series (BALB/c × A/J)F2↔(C57BL × CBA/Ca)F1 × TgTP6.3+/– and new unbalanced series BALB/c↔(C57BL × CBA/Ca)F1 × TgTP6.3+/–.

Previous studies have shown that the composition of chimeric fetuses tends to be strongly positively correlated with other derivatives of the epiblast lineage (such as the amnion and yolk sac mesoderm) but, among different lineages (epiblast vs. hypoblast vs. trophectoderm), correlations are either weaker or absent (West et al. 1984, 1995; James et al. 1993; West & Flockhart, 1994; Tang & West, 2001).Table 3 shows that the composition of the (BALB/c × A/J)F2↔(C57BL × CBA/Ca)F1 × TgTP6.3+/– and BALB/c↔(C57BL × CBA/Ca)F1 × TgTP6.3+/– chimeras also showed these expected relationships between tissues. The compositions of the epiblast derivatives (fetus, amnion and yolk sac mesoderm) were strongly correlated. Comparisons of tissues from different lineages produced weaker correlations, some of which were not significant (the yolk sac endoderm is derived from the hypoblast lineage and the placenta is largely composed of trophoblast cells that are derived from the trophectoderm lineage). This is consistent with previous results for other series of chimeras and again suggests that hemizygous TgTP6.3+/– cells behaved normally and were developmentally neutral.

Table 3.

Correlations, for composition (percentage GPI1-A), between pairs of tissues in the balanced and unbalanced series of E12.5 TgTP6.3+/–↔TgTP6.3–/– chimeras

Balanced series Unbalanced series
Tissues correlated rs P rs P
Within the epiblast lineage
″Fetus vs. amnion 0.871   0.0001 0.853 0.0003
″Fetus vs. YS mesoderm 0.907 < 0.0001 0.861 0.0003
″Amnion vs. YS mesoderm 0.906 < 0.0001 0.926 0.0001
Between lineages
″Fetus vs. YS endoderm 0.653   0.0044 0.283 0.2172 (NS)
″Amnion vs. YS endoderm 0.574   0.0124 0.369 0.1171 (NS)
″YS mesoderm vs. YS endoderm 0.664   0.0038 0.525 0.0259
″Fetus vs. placenta 0.740   0.0013 0.556 0.0154
″Amnion vs. placenta 0.730   0.0015 0.640 0.0066
″YS mesoderm vs. placenta 0.703   0.0022 0.541 0.0217
″YS endoderm vs. placenta 0.684   0.0029 0.324 0.1573 (NS)
Open in a new tab

Abbreviations: rs = Spearman's correlation coefficient; P = probability; NS = not significant (P > 0.05); YS = visceral yolk sac.

Balanced series = (BALB/c × A/J)F2↔(C57BL × CBA/Ca)F1 × TgTP6.3+/– chimeras.

Unbalanced series = BALB/c↔(C57BL × CBA/Ca)F1 × TgTP6.3+/– chimeras.

Discussion

The aim of the present study was to investigate the phenotype and viability of TgTP6.3+/+ homozygotes and TgTP6.3+/– hemizygotes, characterize the expression of the TgTP6.3 transgene in different tissues and evaluate its use as a lineage marker.Pratt et al. (2000) showed that TgTP6.3 tauGFP expression was easy to detect at the cellular level using confocal microscopy on fresh and fixed tissues. One advantage is that, unlike transgenic mice expressing the usual soluble form of GFP, TgTP6.3 tauGFP is highly expressed in axons and blood vessels and so provides a useful new marker for these tissues.Pratt et al. (2000) also performed tissue explants and co-culture experiments, which showed that tauGFP was neither secreted from cells nor passed between cells. Thus, this earlier study showed that TgTP6.3 tauGFP fulfilled four of the six criteria of a good lineage marker noted in the Introduction. It is (1) easy to detect, (2) cell-localized, (3) cell-autonomous and (4) widely expressed. The remaining two criteria are (5) expression should be uniform and stable throughout development and (6) the marker should be developmentally neutral.

In relation to the fourth and fifth criteria, we have extended the range of tissues studied and shown that TgTP6.3 was expressed from the late two-cell stage and expression persisted in most fetal and adult tissues. Although the transgene is widely expressed, it is not truly ubiquitous. GFP expression was not uniform in the adult kidney and was weak or absent in some cell types in the testis and ovary. GFP expression was also very weak or absent in some TgTP6.3+/– cells of the adrenal cortex, which often showed a variegated pattern of radiating stripes. The stripes were similar to those reported by Morley et al. (1996) for 21-hydroxylase/lacZ transgenic mice. In this case the stripes of cells that failed to express lacZ were interpreted as clones of cells that had down-regulated the transgene. Variegated expression patterns have also been reported for other tissues in transgenic mice (Dobie et al. 1997). As tauGFP expression was ubiquitous in pre-implantation embryos the absence of tauGFP in some cells must reflect a down-regulation in certain cell lineages.

The non-uniform, mosaic expression in the adrenal cortex implies that expression in this tissue is down-regulated in some cells during development and therefore the marker must be considered developmentally unstable in this tissue. This precludes use of TgTP6.3+/– hemizygous cells for analysis of the adrenal cortex. It also proved difficult to distinguish TgTP6.3+/– and TgTP6.3–/– cells with confidence in the ovary, testis and kidney, where GFP expression was not uniform. However, tauGFP expression appears to be developmentally stable in other tissues, which indicates that TgTP6.3 meets the fifth requirement of a lineage marker (uniform and stable throughout development) for some cell types.

Confocal microscopy of blastocyst stage TgTP6.3+/–↔TgTP6.3–/– chimeras showed that the two cell populations could be clearly resolved and TgTP6.3 appeared to be a very good marker for use with pre-implantation embryos. The two aggregated embryos showed little mixing in chimeric blastocysts, which agrees with previous studies with other markers (Garner & McLaren, 1974; Dvorak et al. 1995; Everett et al. 2000) and suggests that expression of tauGFP has no significant affect on cell distributions in chimeras at this stage. GFP expression was fairly uniform in many non-chimeric, fetal and adult TgTP6.3+/– tissues. In chimeras, the TgTP6.3+/– cells could be readily distinguished from TgTP6.3–/– cells in these tissues. These chimera studies confirmed that, for many tissues, the TgTP6.3 tauGFP marker fulfilled the first three criteria of a good cell marker as it was easy to detect, cell-localized and cell-autonomous. Moreover, the distribution of TgTP6.3+/– hemizygous cells in the adult liver was very similar to that reported elsewhere using a different genetic marker system (West, 1976), implying that the expression of tauGFP had no obvious effect on cell distributions in chimeric liver, which is relevant to the assessment of developmental neutrality, discussed below.

TgTP6.3+/+ homozygotes died perinatally (between late fetal and weaning age). The reason for the homozygous lethality is unclear but it could be related to the presence of GFP, ectopic expression of tau or the transgenic insertion site. Expression of GFP alone seems an unlikely cause of lethality because many other GFP transgenic lines are viable as homozygotes but tauGFP is qualitatively different from soluble GFP and it is possible that it accumulates to toxic levels in TgTP6.3+/+ homozygotes. Transgenic mice ectopically expressing tau do not necessarily suffer any ill effects but adverse effects have been reported for some transgenic lines expressing high levels of tau protein. These include homozygous fetal lethality and progressive sensorimotor defects and axon degeneration (Ishihara et al. 1999; Spittaels et al. 1999; Probst et al. 2000). Finally, an inappropriate transgenic insertion site may lead to disruption of normal gene expression and deleterious effects. Whatever its cause, the lethality of TgTP6.3+/+ homozygotes makes it unlikely that homozygous cells would be developmentally neutral (criterion 6), and so they are unsuitable for chimera experiments.

Although TgTP6.3+/– hemizygotes were viable and fertile, they were significantly lighter from E14.5 to adulthood. Their small size raised the possibility that TgTP6.3+/– cells might also not be developmentally neutral. Growth and body size could be affected by cell-autonomous effects or by systemic factors. The former are more likely to affect developmental neutrality. Developmental neutrality was investigated by analysing the contribution of hemizygous TgTP6.3+/– cells in mid-gestation chimeric conceptuses from two series of TgTP6.3+/–↔TgTP6.3–/– chimeras. This showed that the overall contribution of hemizygous TgTP6.3+/– cells was similar to non-transgenic cells from (BALB/c × A/J)F2 embryos and was greater than cells from inbred BALB/c embryos, which are known to contribute poorly to chimeras (Mullen & Whitten, 1971; West & Flockhart, 1994; West et al. 1995; Tang & West, 2001). In addition, the distributions of the compositions of the individual fetuses and different extra-embryonic tissues and the quantitative relationships between tissues were similar to those reported for other series of chimeras, implying that expression of the tauGFP transgene in hemizygous TgTP6.3+/– cells did not significantly affect the tissue composition of the chimeras. These results imply that hemizygous TgTP6.3+/– cells were developmentally neutral in chimeras, at least until E12.5. Hemizygous TgTP6.3+/– cells also contributed well to postnatal chimeric tissues. Overall the results suggest that they are likely to be developmentally neutral and so probably fulfil the sixth criterion required of a good lineage marker.

In conclusion,Pratt et al. (2000) showed that the TgTP6.3 transgene provides an excellent marker of neural processes and subcellular microtubule structures in some living and fixed tissues and that tauGFP had advantages over soluble forms of GFP.Pratt et al. (2000) also showed that expression of tauGFP in hemizygous TgTP6.3+/– fulfilled many of the criteria of a good lineage marker. This study has shown that expression of TgTP6.3 was uniform (and therefore developmentally stable) in many tissues. It also provides evidence that TgTP6.3 is developmentally neutral in hemizygous TgTP6.3+/– cells and that these cells contribute well to chimeric tissues and mix normally with non-transgenic cells. TgTP6.3 fulfils the critical requirements of a lineage marker for at least some tissues and, although it is unlikely to be the most appropriate marker for all tissues or for all experimental situations, it provides a useful new addition to the lineage markers currently available for chimera and cell mixing experiments. It proved to be a good cellular marker for chimera studies in a range of adult and fetal tissues, and is particularly well suited for highlighting axons and blood vessels and for use with chimeric pre-implantation embryos.

Acknowledgments

We thank Denis Doogan, Maureen Ross, Duncan McNeil and Jim Macdonald for expert mouse husbandry. We are grateful to the Wellcome Trust (grants 059904/JS and 052578/Z/97/Z), the College of Medicine and Veterinary Medicine, University of Edinburgh (PhD studentship for G.E.M.) and the University of Edinburgh Moray Endowment Fund for financial support.

References

  1. Collinson JM, Quinn JC, Buchanan MA, et al. Primary defects in the lens underlie complex anterior segment abnormalities of the Pax6 heterozygous eye. Proc. Natl Acad. Sci. USA. 2001;98:9688–9693. doi: 10.1073/pnas.161144098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Court FA, Sherman DL, Pratt T, et al. Restricted growth of Schwann cells lacking Cajal bands slows conduction in myelinated nerves. Nature. 2004;431:191–195. doi: 10.1038/nature02841. [DOI] [PubMed] [Google Scholar]
  3. Dobie K, Mehtali M, McClenaghan M, Lathe R. Variegated gene expression in mice. Trends Genet. 1997;13:127–130. doi: 10.1016/s0168-9525(97)01097-4. [DOI] [PubMed] [Google Scholar]
  4. Dvorak P, Yoshiki A, Dvorakova D, Flechon JE, Kusakabe M. Cell mixing during the early development of mouse aggregation chimera. Int. J. Dev. Biol. 1995;39:645–652. [PubMed] [Google Scholar]
  5. Everett CA, Stark MH, West JD, Davidson D, Baldock RA. Three-dimensional reconstruction of tetraploid↔diploid chimaeric mouse blastocysts. J. Anat. 2000;196:341–346. doi: 10.1046/j.1469-7580.2000.19630341.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Garner W, McLaren A. Cell distribution in chimaeric mouse embryos before implantation. J. Embryol. Exp. Morph. 1974;32:495–503. [PubMed] [Google Scholar]
  7. Hadjantonakis AK, Gertsenstein M, Ikawa M, Okabe M, Nagy A. Generating green fluorescent mice by germline transmission of green fluorescent ES cells. Mech. Dev. 1998;76:79–90. doi: 10.1016/s0925-4773(98)00093-8. [DOI] [PubMed] [Google Scholar]
  8. Hadjantonakis AK, Dickinson ME, Fraser SE, Papaioannou VE. Technicolour transgenics: imaging tools for functional genomics in the mouse. Nature Rev. Genet. 2003;4:613–625. doi: 10.1038/nrg1126. [DOI] [PubMed] [Google Scholar]
  9. Ikawa M, Kominami K, Yoshimura Y, Tanaka K, Nishimune Y, Okabe M. Green fluorescent protein as a marker In transgenic mice. Dev. Growth Differ. 1995;37:455–459. doi: 10.1046/j.1440-169X.1995.t01-2-00012.x. [DOI] [PubMed] [Google Scholar]
  10. Ikawa M, Yamada S, Nakanishi T, Okabe M. ‘Green mice’ and their potential usage in biological research. FEBS Lett. 1998;430:83–87. doi: 10.1016/s0014-5793(98)00593-6. [DOI] [PubMed] [Google Scholar]
  11. Ishihara T, Hong M, Zhang B, et al. Age-dependent emergence and progression of a tauopathy in transgenic mice overexpressing the shortest human tau isoform. Neuron. 1999;24:751–762. doi: 10.1016/s0896-6273(00)81127-7. [DOI] [PubMed] [Google Scholar]
  12. James R, Flockhart JH, Keighren M, West JD. Quantitative analysis of mid-gestation mouse aggregation chimaeras: non-random composition of the placenta. Roux's ArchDevBiol. 1993;202:296–305. doi: 10.1007/BF00363218. [DOI] [PubMed] [Google Scholar]
  13. Lyon MF. X-ray induced dominant lethal mutations in male guinea-pigs, hamsters and rabbits. Mutat. Res. 1970;10:133–140. doi: 10.1016/0027-5107(70)90158-2. [DOI] [PubMed] [Google Scholar]
  14. McLaren A. Mammalian Chimaeras. Cambridge: Cambridge University Press; 1976. [Google Scholar]
  15. Morley SD, Viard I, Chung BC, Ikeda Y, Parker KL, Mullins JJ. Variegated expression of a mouse steroid 21-hydroxylase/β-galactosidase transgene suggests centripetal migration of adrenocortical cells. MolEndocrinol. 1996;10:585–598. doi: 10.1210/mend.10.5.8732689. [DOI] [PubMed] [Google Scholar]
  16. Mullen RJ, Whitten WK. Relationship of genotype and degree of chimerism in coat color to sex ratios and gametogenesis in chimeric mice. JExpZool. 1971;178:165–176. doi: 10.1002/jez.1401780203. [DOI] [PubMed] [Google Scholar]
  17. Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y. ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett. 1997;407:313–319. doi: 10.1016/s0014-5793(97)00313-x. [DOI] [PubMed] [Google Scholar]
  18. Pratt T, Sharp L, Nichols T, Price DJ, Mason JO. Embryonic stem cells and transgenic mice ubiquitously expressing a tau-tagged green fluorescent protein. Dev. Biol. 2000;228:19–28. doi: 10.1006/dbio.2000.9935. [DOI] [PubMed] [Google Scholar]
  19. Probst A, Gotz J, Wiederhold KH, et al. Axonopathy and amyotrophy in mice transgenic for human four-repeat tau protein. Acta Neuropathol(Berlin) 2000;99:469–481. doi: 10.1007/s004010051148. [DOI] [PubMed] [Google Scholar]
  20. Quinn P, Barros C, Whittingham DG. Preservation of hamster oocytes to assay the fertilizing capacity of human spermatozoa. J. Reprod. Fert. 1982;66:161–168. doi: 10.1530/jrf.0.0660161. [DOI] [PubMed] [Google Scholar]
  21. Reese BE, Necessary BD, Tam PPL, Faulkner-Jones B, Tan SS. Clonal expansion and cell dispersion in the developing mouse retina. Eur. J. Neurosci. 1999;11:2965–2978. doi: 10.1046/j.1460-9568.1999.00712.x. [DOI] [PubMed] [Google Scholar]
  22. Spittaels K, Van den Haute C, Van Dorpe J, et al. Prominent axonopathy in the brain and spinal cord of transgenic mice overexpressing four-repeat human tau protein. Am. J. Pathol. 1999;155:2153–2165. doi: 10.1016/S0002-9440(10)65533-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tang P-C, West JD. Size regulation does not cause the composition of mouse chimaeras to become unbalanced. IntJDevBiol. 2001;45:583–590. [PubMed] [Google Scholar]
  24. Tarkowski AK. Mouse chimaeras developed from fused eggs. Nature. 1961;190:857–860. doi: 10.1038/190857a0. [DOI] [PubMed] [Google Scholar]
  25. Tyas DA, Pratt T, Simpson TI, Mason JO, Price DJ. Identifying GFP-transgenic animals by flashlight. Biotechniques. 2003;34:474–476. doi: 10.2144/03343bm04. [DOI] [PubMed] [Google Scholar]
  26. West JD. Patches in the livers of chimaeric mice. J. Embryol. Exp. Morph. 1976;36:151–161. [PubMed] [Google Scholar]
  27. West JD. Cell markers. In: Douarin NL, McLaren A, editors. Chimeras in Developmental Biology. London: Academic Press; 1984. pp. 39–63. [Google Scholar]
  28. West JD, Bücher T, Linke IM, Dünnwald M. Investigation of variability among mouse aggregation chimaeras and X chromosome inactivation mosaics. J. Embryol. Exp. Morph. 1984;84:309–329. [PubMed] [Google Scholar]
  29. West JD, Flockhart JH. Genotypically unbalanced diploid↔diploid foetal mouse chimaeras: possible relevance to human confined mosaicism. Genet. Res. 1994;63:87–99. doi: 10.1017/s0016672300032195. [DOI] [PubMed] [Google Scholar]
  30. West JD, Flockhart JH, Kissenpfennig A. A maternal genetic effect on the composition of mouse aggregation chimaeras. Genet. Res. 1995;65:29–40. doi: 10.1017/s0016672300032985. [DOI] [PubMed] [Google Scholar]
  31. Ying QL, Nichols J, Chambers I, Smith A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell. 2003;115:281–292. doi: 10.1016/s0092-8674(03)00847-x. [DOI] [PubMed] [Google Scholar]
  32. Zernicka-Goetz M, Pines J, Hunter SM, et al. Following cell fate in the living mouse embryo. Development. 1997;124:1133–1137. doi: 10.1242/dev.124.6.1133. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Anatomy are provided here courtesy of Anatomical Society of Great Britain and Ireland

RESOURCES