Boatright et al.
Jeffrey H. Boatright, Eleanore A. Gross, John W. Peoples, and John M.
Emory Eye Center, Emory University School of Medicine, Atlanta, GA
Correspondence should be addressed to:
Jeffrey H. Boatright
Lab B-5500, Emory Eye Center
1327 Clifton Rd.
Atlanta, GA 30322
Keywords: cell culture, chick embryo, incubator, embryogenesis
Our laboratory recently developed a primary embryonic chicken cell
culture system for gene promoter activity studies (1). To avoid stage-specific
variables in promoter function, we begin all experiments on the same embryonic
day of development. Since fertilized eggs are delivered only once a week, the
rate of experimentation is limited. We increased this rate by taking advantage
of the temperature dependency of chick embryogenesis. Normal chick
embryogenesis proceeds to hatching 21 days following fertilization (2) if eggs
are incubated at 37deg.C. Development is arrested if the eggs are held at
15.5deg.C, but resumes when the eggs are brought to 37deg.C. By transferring
eggs stored at 15.5deg.C to 37deg.C on consecutive days, then allowing
embryogenesis for a set duration, we can conduct experiments daily using
embryos that are at the same developmental stage.
This strategy requires an incubator that maintains 15.5deg.C. Several vendors
sell such incubators, but they are expensive: between $1700 and $3500. We
assembled an alternative from a used freezer and a thermostatic power
interrupter (thermostat) for less than $50.00. The heart of the system is the
inexpensive thermostat made available to us through the homebrewing market
(Table I). Successful beer brewing requires constant and precise temperatures
warmer than those provided by typical refrigerators. To achieve this,
homebrewers combine old refrigerators with thermostats that tightly regulate
fermentation temperatures (4, 5).
MATERIALS AND METHODS
Assembly of a low temperature incubator
The internal thermostat of the
cooling unit is set to "coldest" position. The separate external thermostat is
set to 15.5deg.C (60deg.F). The cooling unit's power cord is plugged into the
external thermostat, and the thermostat into the wall outlet. The thermostat's
temperature probe is suspended inside the refrigerator, dangling free
(Figure 1). In less than 2 hours, the newly-constructed low-temperature
should be temperature-stable and ready to use (3).
Our cooling unit is a used Sears Kenmore Model 8 chest freezer of 8.3
ft3 internal capacity. Any refrigerator or freezer should work. If
a self-defrosting refrigerator is used, disable the defrost clock. It is not
needed for our purposes, and compared to other components, has a high failure
rate. The clock is usually a small black box with a knob and two wires
connected. It commonly is found on the underside of the refrigerator near the
rear. Bypass the clock by disconnecting the wires from the box and soldering
There are several thermostats available from brew or refrigeration supply
houses (Table I). We use the Hunter Energy Monitor (Amber Waves, Atlanta, GA).
Incubator temperatures were initially recorded on a Dickson Model TH4-7C
temperature recorder (The Dickson Company, Addison, IL). Though the
differential between turn-on and turn-off for our thermostat is given as -
1deg.C and +1.5deg.C, our incubator initially fluctuated much more,
overshooting as much as 4.3deg.C below the target temperature. Adding a small
fan to aid air circulation kept temperatures from varying more than +/-0.5deg.C
(data not shown). Despite the air-tight seal of the freezer lid and the 10
dozen eggs contained within, CO2 levels in the incubator, as
measured with a Fyrite test kit (Bacharach Instrument Company, Pittsburgh, PA)
were indistinguishable from ambient levels (data not shown).
Arresting embryogenesis for up to a week has no detectable effect on
development. Embryos incubated at 37deg.C for varying numbers of days following
6 days of 15.5deg.C arrest appear no different than those not arrested (Figure
2). At late E6 (6 days of incubation at 37deg.C; Carnegie Stage 19 or Hamburger
and Hamilton Stage 30 ), both sets of embryos have distinct digital rays,
developing egg teeth, and emerging eyelids (Figure 2 top). Both sets of E10
embryos (Carnegie Stage 22-23 or Hamburger and Hamilton Stage 35-36) have
developed digits with minimal webbing, eyelids that are beginning to close, and
the emergence of claws (Figure 2 bottom).
Importantly for our eventual use of the embryos, promoter activity is
unaffected by short developmental arrest. For these experiments, retinal cells
from E10 embryos (i.e., incubated at 37deg.C for 10 days) with or without a
previous 15.5deg.C developmental arrest of 6 days were transfected with plasmid
vectors containing the chloramphenicol acetyltransferase (CAT) gene linked to a
trunctated version of the promoter of the murine interphotoreceptor
retinoid-binding protein (IRBP). CAT activity was assayed subsequently and used
as a measure of promoter activity (1). Promoter activity was statistically
indistinguishable between cultures derived from arrested or non-arrested
embryos (925 +/- 23 vs 945 +/- 66 x 10-5 units
respectively. Data are mean CAT activities +/- SEM; N = 4 per group,
P > 0.5). This suggests that short arrest periods have little effect
on this measure of functionality.
Prolonged 15.5deg.C arrest decreases embryo viability. Arrest periods of up to
7 days have little effect on embryo survival following a subsequent 10 day
incubation at 37deg.C (Figure 3). However, embryo survival drops
precipitously with increasing arrest periods. Survival is below 50% with 10-12
days of 15.5deg.C developmental arrest. No embryos developed if arrested for
more than 22 days (Figure 3).
Though 15.5deg.C is the optimal temperature for lager fermentation (3, 4) and
chick embryogenesis arrest, we suggest maintaining separate incubators for eggs
- Boatright, J. H., R. Buono, J. Bruno, R.K. Lang, J.-S. Si, T. Shinohara, J.
W. Peoples and J.M. Nickerson. 1996. The 5' flanking regions of mouse IRBP and
arrestin have promoter activity in primary embryonic chicken retina cell
cultures. Exp. Eye Res. In press.
- Butler, H. and B.H.J. Juurlink. 1987. An Atlas for Staging Mammalian and
Chick Embryos. CRC Press, Boca Raton. FL.
- O'Connor, K. The in-laws' refrigerator. Brewing Techniques,
November/December, 1993: 45-46.
- Walz, G. 1995. Brewtek ferm temp thermostat. Zymergy, Fall, 1995: 94-95.
© 1996 Epress Inc.