It has been half a century since the first studies on cell biology. During this time, it was shown that cells consist of a membrane made
of lipid-polymer complexes comprising proteins and carbohydrates complexed with lipids. Further studies on cell biology have been
based according to this understanding of the structure of the cell membrane. No reports of a cell membrane associated with DNA have
been reported. I have been studying the preparation of artificial cells and have demonstrated the formation of cells (named DNA crown
cells) containing a membrane comprising lipid-DNA. I have prepared DNA crown cells using a known lipid (monolaurin) in vitro and
examined whether these cells self-replicate in vivo. Here, I discuss these successful procedures. First, aggregates of sphingosine-DNA
were prepared by the addition of adenosine-monolaurin to sphingosine-DNA. Next, DNA crown cells were constructed by the addition
of monolaurin to the sphingosine-DNA aggregates in vitro. Finally, I demonstrated that these cells can self-replicate upon incubation in
Artificial Cells, DNA Crown Cells, Sphingosine-DNA Adenosine-Lipid, Aggregates of Sphingosine-DNA, Self-Replication
Artificial cells, including liposomes, nanoparticles, and
microcapsules, have been studied since the 1960s and have led
to impressive achievements in the generation of artificial cells
(Zhang et al, 2008; Lin et al. 2013; Kuruma et al. 2009). However,
to date, artificial cells which can replicate autonomously have not
Recent work on artificial cells has focused on cell division or
replication (Noireaux et al, 2011). I have developed a method to
prepare artificial cells (Inooka, 2012; Inooka, 2016, Ref.5). The
artificial cells are prepared by incubating in egg-white artificial
cell seeds comprising sphigosine (Sph), DNA, and components
that bind Sph-DNA, such as adenosine or uridine (Inooka, 2016;
Ref.7). I previously reported (Inooka, 2016, Ref; 8) that Sph-
DNA aggregated with adenosine-lipids contained in egg-white
to generate DNA crown cells. Here, I attempted to prepare DNA
crown cells using a known and commercially available lipid,
monolaurin. First, adenosine-lipids were prepared, then DNA
crown cells were constructed using the resulting aggregates of
Sph-DNA. The procedures for synthesizing DNA crown cells are
Materials and Methods:
Sph (Sigma, USA), DNA (Escherichia coli strain B, Sigma, USA),
Adenosine (Sigma, USA and Wako, Japan), monolaurin (Tokyo
Kasei, Japan), and white Leghorn eggs purchased from a market.
Preparation of the compound from by mixing adenosine and
monolaurin (A-M) A case to prepare A-M is as follows: 0.4 ml (0.1
M) monolarurin was added to 0.4 ml (0.1M) of adenosine solution.
After mixing, 0.15 ml of ethanol was added to the mixture and
the precipitate was collected and dried. The A-M precipitate was
resolved in 1.0 ml distilled water and used. Effect of compound
(A-M) on Sphingosine-DNA To examine the effect of A-M on
Sph-DNA, Sph (90μl, 10mM) was added to 40μl/μl of DNA
(1.7μg/μl). After heating the mixture, A-M solution (50μl) was
added., then one drop of ethidium bromide solution was added to
one drop of the Sph-DNA-A-M mixture. Then , a drop was placed
on a glass slide and observed using phase contrast microscopy and
Synthesis of DNA crown cells:
To synthesize DNA crown cells, Sph (90μl, 10mM) was added
to 40μl of DNA (1.7 μg/g/μl). After heating the mixture, A-M
solution (50μl) was added and monolaurin solution (50μl, 0.1M) was added to the Sph-DNA-A-M mixture. After mixing, the cells
were observed as described above. Artificial cell generation using a
mixture of Sph-DNA-A-M solution and transplantation To generate
artificial cells using the Sph-DNA-A-M mixtures, 90μl of Sph (10
mM) was added to 40μl of DNA (1.7μg/μl). After heating, A-M
solution (100μl) was added to the Sph-DNA mixture. A hole was
drilled in the shell of an edible white Leghorn egg and 0.25~0.5 ml
of the mixture was injected into the egg white (albumin). Each egg
was then incubated for 7 days at 37℃, and a portion of each egg
white was removed. About 1 ml of egg albumin was added to 10
ml of Dulbecco’ Modified Eagles Medium containing 10% bovine
serum (DMEM) and cultured for 2 days at 37℃. Aggregates of
artificial cells were found at the bottom of the culture medium.
A drop of the aggregates was placed on a glass slide and the
integrity of the artificial cells was observed using phase contrast
microscopy and fluorescence microscopy. The artificial cells
were transplanted by inoculating a sample of egg-white (0.5ml)
containing the cells into fresh egg-white weekly. Also, 2 ml of
egg-white containing cells was incubated in 10 ml of DMEM and
incubated at 37℃ for 2 days. Transplantation was carried out until
the fifth generation.
Result and Discussion:
Effect of A-M on Sphingosine-DNA To prepare adenosine-lipid
compound, the lipid used was monolaurin, based on previous
experiments (Inooka, 2016, Ref.8). monolaurin solution was
added to adenosine solution. After mixing, ethanol was added,
the precipitate was collected and dried, then, the A-M precipitate
was resolved in distilled water Sph (90μl,10mM) was added to
40 μl of DNA (1.7μg/μl), heated, then the A-M solution (50μl)
was added. One drop of the Sph-DNA-A-M mixture was placed on
a glass slide and observed using phase contrast and fluorescence
Two types of aggregates were observed. One was mucoid-type,
as shown in Fig. 1a, and the other was crystal-type, as shown in
Aggregates of sphingosine (Sph)-DNA with adenosinemonolaurin
(A-M). Sph was added to DNA, then A-M was
added to the Sph-DNA mixture. Two types of aggregates formed:
mucoid-type, shown in Fig. 1a, and crystal-type, shown in Fig.
1b.Scale bar 50 μm.
Typical crystal-type of aggregates was shown in Fig1c. The sample
was stained with ethidium bromide and Russert light was observed
on the surfaces of the crystal aggregates under fluorescence
microscopy (Fig. 1d), suggesting that the surface contains DNA.
Scale bar 50 μm. Fig. 1c and Fig. 1d are the same field of view.
Construction of DNA crown cells.
Sph was added to DNA. After mixing, A-M was added to Sph-
DNA mixture, then monolaurin was added to the Sph-DNA-A-M
mixture, stained with ethidium bromide, and smeared on a glass
slide. The images in a) and b), and in c) and d), are the same field
a) Phase contrast microscopy observation, showing cells of various
sizes Scale bar 50μm
b) Russert light is observed on the surfaces of the particles under
fluorescence microscopy, indicating that DNA is present on the
surface of the cells. Scale bar 50μm.
c) A typical large particle observed using fluorescence microsc py
is shown in Fig. 2c. Russet light was observed on the wall of the
cell, indicating that the wall contains DNA. Scale bar 20 μm.
d) A typical small cell observed using fluorescence microscopy
is shown in Fig. 2d. Russet light was observed on the particle,
indicating that particles contain DNA.
Scale bar is 20μm.
Fig. 1b. Typical crystal aggregates are shown in Fig. 1c. Russert
light was observed on the edge of the crystal using fluorescence
microscopy (Fig. 1d), suggesting that the edge contains DNA. This
may be explained as follows: The addition of DNA to Sph results
in the formation of fibrous assemblies of Sph-DNA (Inooka, 2000;
Inooka, 2014). The assembly may form particulates upon heating.
The assembly comprising particles aggregated upon the addition
of A-M solution. Synthesis of DNA crown cells from aggregates
of Sph-DNA-A-M. A solution of monolaurin was added to the
Sph-DNA-A-M aggregates and one drop of the mixture was
placed on a glass slide and observed using both phase contrast and
Round cells of various sizes were observed, as shown in Fig.
2a. Russert light was observed on the surface of each cell under
fluorescence microscopy, as shown in Fig. 2b, indicating that DNA
crown cells had formed. A typical large cell as observed using
fluorescence microscopy is shown in Fig. 2c and a typical small
cell is shown in Fig. 2d. Russert light is clearly observed on the
surfaces of both cells. The shrinkage of large cells may result in the
formation of cells of various sizes.
Artificial fifth generation cells generated using mixtures of Sph-DNA-A-M. The Sph-DNA-A-M mixtures were incubated in
egg-white for 7 days, then the egg-white contained artificial cells were transplanted into a new egg. This transplantation was carried out
every 7 days for five generations. Fifth generation egg-white (2 ml) was cultivated in Dulbecco’s Modified Eagle’s Medium containing
10% bovine serum for 2 days. Aggregation of the artificial cells was evident at the bottom of the culture tube. Aggregates were stained
with ethidium bromide and smeared on a glass slide, then observed using phase contrast and fluorescence microscopy. A phase contrast
micrograph is shown in Fig.3a. Three clear cells (arrow) were observed. Russet light was observed on the surfaces of the cells under
fluorescence microscopy, showing that the surfaces of the cells were covered with DNA (Fig. 3b).
Scale bar 20μm. Fig.3a and Fig.3b are the same field of view.
These large and small cells could be formed by the aggregation of
crystal-type and mucoid-type cells. The generation of artificial cells
from Sph-DNA-A-M mixtures and transplantation of DNA crown
cells in vivo. Sph-DNA-A-M mixtures were prepared as described
in Step 3. Each mixture was injected into the white (albumin) of
individual eggs. After incubation at 370C for 7 days, 0.5 ml of eggwhite
potentially containing artificial cells was inoculated into
fresh egg-white weekly for five weeks. One week’s incubation is
defined as one generation. For each generation, 2 ml of egg-white
containing cells was incubated in 10 ml of DMEM and incubated
at 370C for 2 days. Aggregates at the bottom of the culture medium
were observed. The structural integrity of the artificial cells after 5
generations was assessed by phase contrast microscopy (Fig. 3a)
and fluorescence microscopy (Fig. 3b). Three DNA crown cells
(arrows) were clearly observed, indicating that DNA crown cells
replicated in egg-white.
Thus, I have succeed in preparing DNA crown cells using the
purified reagents sphingosine, DNA, adenosine and monolaurin.
I have also demonstrated (Inooka, 2016; Ref.7) that artificial cells
are generated using Sph-DNA and nucleosides, including uridine.
Therefore, mixtures prepared with combinations of nucleosides
and monolaurin may aggregate Sph-DNA. Moreover, compounds
prepared using combinations of nucleosides and lipids related
to monolaurin may also form aggregates of Sph-DNA and thus
construct DNA crown cells. Here, I used DNA from Escherichia
coli. These cells can be prepared using DNA from other sources
This means that various DNA crown cells consisting of different
components could be prepared by combining nucleosides, lipids
and DNA. The current method for preparing DNA crown cells can
be performed easily. Cells whose membrane consists of DNAlipid
have not previously been reported. This method is readily
accessible to anyone and may provide new findings in the life
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