Laser-assisted Thermal-expansion Microinjection

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					                 Laser-assisted Thermal-expansion Microinjection
                 Tetsuya Higashiyama
                 Graduate School of Science, Nagoya University
 Here we report a novel microinjection method designated as "Laser-assisted Thermal-expansion Microinjection" (patent application number (2004) 361448). This
 method generate extremely high pressure so that we could use capillaries with fine tips around 0.1µm to inject materials. Thus, precise injection was possible, and
 damage to cells and cell-components was drastically reduced. High pressure at the tip enabled us to inject plasmid DNAs into more than 50 plant cells by using a
 single capillary, although injection into the plant cell (having cell wall) by using such a fine tip is impossible by conventional methods. Advantages and examples of
 this simple method will be shown and discussed.

[Introduction]                                                                                                                   [Results & Discussion]
We previously succeeded in vitro pollen
tube guidance to the female gametophyte,
by using Torenia fournieri that has a naked
female gametophyte (Higashiyama et al.,
Plant Cell, 1998). Typical chemotropism is
observed in this system (Fig. 1, top panels).
We identified the “synergid cell” on the side
of egg cell as the source of the attractant,
by laser ablation experiments (Higashiyama
e t a l ., S ci e nce , 2 00 1 ; Fig. 1 , b o tt om
panels). Although chemical of the
a tt ra cta n t h a s b ee n u n kn o w n, i t w a s
suggested that the attractant might be a
molecule synthesized in the
synergid cell. We are now
tryin g to establish cDNA
library of the synergid cell.
We are planning RNAi
analysis using the EST data                                                                                                           Fig.2 Diagram of the Laser-assisted Thermal-expansion Microinjection (LTM)
of it. In this study, we try to
develop novel injection                                                                                                              Heat-induced expansion of liquid in a sealed capillary generate
system to introduce materials Fig.1 Pollen tube attraction by the synergid cell                                                      high pressure, which enables injection by capillaries with the tip-
i n t o t h e s y n e r g i d c e l l (A) to (D): A pollen tube chasing a manipulated female gametophyte.                            diameter around 0.1µm (Knoblauch et al., Nature Bitotech., 1999).
e f f i c i e n t l y, a l t h o u g h i t i s Bottom panels: laser ablation experiment showing that the synergid cell in            As the heat source, we chose laser beam which enabled us to
d i f f i c u l t b y c o n v e n t i o n a l the female gametophyte is the source of the attractant.                                directly heat hydrophobic liquid containing laser absorber though
methods.                                                                                                                             the capillary glass and to precisely control the injection flow.

               Instead of air pressure or oil pressure through silicone tube,
               IR laser is irradiated to the capillary through optical fiber.
               Injection flow can be precisely controlled by adjusting output
               level of the laser power. To avoid back-filling of the
               capillary, output level is gradually increased but not
               decreased in a series of injection.

                  Fig.3 Equipments of LTM
                  The capillary holder is connected to Nd:YAG laser with optical fiber.

Due to high pressure (approximately                                                     20                                      By using LTM, we could
10 atm per 1 ), it was possible to use                                                                                          readily inject materials
capillaries with the tip around 0.1µm.                                                                                          into the female gameto-
Fig. 4 shows comparison of capillary                                                                                            phytic cells and their
for conventional method and LTM.                                                                                                components of Torenia,
LTM capillaries were made by Sutter                                                     10                                      as we aimed. LTM will
P97 puller. Such capillaries with fine                                                                                          allow us to perform
tips could not be used for conven-                                                                                              novel approach, inclu-
tional method (oil pressure) at all.                                                      5
                                                                                                                                ding RNAi analysis in
However, we could use the capillaries                                                                                           the synergid cell by
for LTM without clogging the tip. More                                                                                          directly injecting double
than 5 0 toba cco BY-2 cells were                                                             thick tip fine tip   fine tip
                                                                                                                                stranded RNA.
                                                                                                Conventional         LTM
injected in a single capillary.                                                                    method

Fig.4 Capillaries with fine tips around 0.1µm for LTM                                                                                                                                         10µm                          20µm
SEM images of tips of conventional (~1.3µm; top on the left) and LTM (~0.1µm; bottom on the left)
capillaries and efficiency of injection with these capillaries (right) are shown. To examine the efficiency,                    Fig.5 LTM to the naked female gametophyte of Torenia
plasmids with Alexa-dextran 10k were injected into tobacco BY-2 cells, as in the case of Fig. 5, by using a                     Injection of plasmids with Alexa 546-dextran 10K into the central cell nucleus (left) and the
single capillary (up to 20 cells).                                                                                              synergid cell cytosol (right) is shown.

Nuclear injection drastically increases                                                                                         To test the ability of precise
the transformation rate than the case of                                                                                        injection by LTM, we further
cytosolic injection. However, nuclear                                                                                           tried more smaller objects.
injection in plant cells is difficult, and                                                                                      St r i k i n g l y, i n j e c t i o n o f
reports are still rare. We found that                                                                                           fluorochromes into yeasts was
onion epidermal cells were good system                                                                                          possible as shown in Fig. 7.
t o s e t u p t h e c o n d i t i o n o f n u cl e a r                                                                          This suggests that LTM could
i n j e c t i o n u s i n g LT M , a l t h o u g h , b y                                                                        be applied to a lot of kinds of
conventional methods, rigid cell wall                                                                                           cells, including prokaryotic
hamper the injection. As shown in Fig. 6,                                                                                       cells and organelle.
some population of injected cells were                                                                                             We are preparing for sell-
transformed and expressed GFP as                                                                                                ing products and looking for
tr a n s ie n t e xp r e s si o n . D e sp it e t h e                                                                           co l l a b o r a t o r s t o t e s t LT M
successful injection and high viability of                                                                                      toward various types of cells
cells, transformation rate is still low                                                                                         and organelle. Please contact                                                              10µm
(below 10%). Investigation of good                                                                                              us if you are interested in LTM
condition for plant transformation by                                                                                    50µm   (
microinjection is now on progress.
 Fig.6 Transformation of onion epidermal cells by nuclear injection                                                               Fig.7 Injection into yeasts
 Top panels: an epidermal cell just after nuclear injection (LTM) of plasmids with Alexa 546-dextran 10K. Bottom                  A diploid cell of yeast W303 at the log phase was hold by the aspiratory capillary (“left hand”
 panels: a transformed epidermal cell 16 hours after injection, expressing NLS+GFP (bottom right). Arrows and                     as seen in the top panel) and was injected with Alexa 488-phalloidin using LTM.
 arrow heads indicate nuclei and insertion marks of the capillary, respectively.