mode of transmission in glofish1
Post on 07-Aug-2015
19 Views
Preview:
TRANSCRIPT
Myers 1
Christopher Myers
Augsburg College
Genetics Research Project
Mode of Transmission in GloFish
Abstract
This study was designed to study the mode of transmission in a mutant type
of Zebrafish known as GloFish. More specifically the green fluorescent protein
(GFP) or glow transgene was observed to determine how it behaves regarding
heritability. We applied a reciprocal cross of male vs. female, mutant type vs. wild
type to produce an F1 generation of both crosses to a particular embryonic
developmental stage to examine if the GFP gene is expressed and observable if
present. By this method we were able to determine that the gene was autosomal
dominant and not sex-‐linked, autosomal recessive, codominant or incompletely
dominant. It was also found that though sex-‐linkage was not present an apparent
maternal effect was. Though previous studies with these fish have been done at
Augsburg College before, our experiment was novel in the way that it was done to
determine how, and if, sex played a role in the mode of transmission.
Introduction
Over the past several decades Zebrafish (Danio rerio) have been extensively
studied from several different types of scientists to students both in colleges and
Myers 2
high schools around the globe. This is due to the fact that these fish are very
inexpensive, can tolerate a reasonable amount of stress, produce a lot of eggs,
produce these eggs reliably, and allow observers to watch embryo development that
is comparable to other species (Zebrafish FAQs). This embryonic development is
relatively quick and is easily observable under a decent microscope. Because the
embryos can be seen so easily and gene expression occurs quickly these fish have
become ideal research organisms; and though they are vastly different from
humans, and other mammals, their embryonic development is still quite similar to
all vertebrates that seem to follow a developmental program that is evolutionarily
conserved (Kimmel et al., 2012).
Recently the Zebrafish have been genetically altered into several different
strains of fish that will glow different colors due to an inserted transgene. These fish
are known as GloFish and are readily commercially available in the pet market as
well as offer significant viability in
laboratory experiments. Under normal
light these fish appear to be brightly
colored, however, when they absorb
certain wavelengths of light they are able
to fluorescence (glow). Due to their vast
array to explore genetic concepts at the
fundamental level, this organism was
chosen for our experiment in order to look at how this transgene is inherited (Vick
et al., 1995).
Figure 1. Different colors of GloFish http://www.thatpetplace.com/glofish-‐danios
Myers 3
By looking at the main manufacturers and distributor’s of GloFish website
GloFish.com several different types of GloFish have been patented and trademarked.
The different types/colors are bright red, green, orange-‐yellow, blue, and purple
(Figure 1). In this experiment electric green was used, which appear to be yellow
under normal light. The source for the transgene inserted into the green GloFish
comes from the Aequorea victoria jellyfish (GloFish® FAQ).
Methods
Mutant type GloFish (8) and wild type Zebrafish (6) were obtained by
Professor Beckman from a pet store and initially all placed in a large single tank.
This was done in order to relieve stress on the fish and ease the acclamation
process. The tank was located in an incubator at 28.5 °C with a light-‐dark cycle.
Around a week and a half before the fish were mated, both mutant and wild types
were sexed and separated accordingly: all males in one tank and all females in
another. The night before the intended mating, two mating tanks were filled with
50/50 mix of water from the tanks in which both the female and male fish were
taken from. The first mating tank included one female GloFish and two male
Zebrafish. The second mating tank included one female Zebrafish and two male
GloFish. These tanks were then placed back into the incubator and the fish were fed
again to make them comfortable.
Eggs were collected the following morning, roughly 4 hours post fertilization,
from each mating tank via pipettes and microscopes and placed into two labeled
petri plates containing embryo water made up in the lab (Figure 2). Any eggs that
Myers 4
showed abnormalities were discarded from the samples. The following day the eggs
were examined again for any other abnormalities or developmental problems; any
that were found were discarded. Around 50 hours post fertilization all of the
healthy embryos were mounted (five embryos per slide) and observed under a
florescence microscope with blue light settings (Figure 2). These embryos were
then scored on a positive/negative scale on if the expression of GFP was present by
observable fluorescence.
Figure 2. Example of egg 4 hours post fertilization and embryo 48 hours post fertilization. Eggs were collected 4 hours post fertilization and embryos were examined for presence of GFP 50 hours post fertilization. Photo credit (Kimmel et al., 1995).
Results and discussion
The female Glofish produced a much larger sample size of 39 viable eggs
compared to the 10 viable eggs produced by the female Zebrafish. The female
Zebrafish had many eggs that contained abnormalities and many had to be
discarded of. Out of the 39 embryos produced by the maternal mutant type GloFish
and paternal wild type Zebrafish all of them expressed the GFP gene. For the
maternal wild type Zebrafish and paternal wild type GloFish 4 out of the 10
expressed the GFP gene (Table 1).
Table 1. GFP presence amongst both embryo samples.
Myers 5
It was also observed that in all of the maternal Glofish embryos (39)
fluorescence was observed throughout most of the embryo’s tissues (Figure 3),
however, in the maternal Zebrafish embryos (4) fluorescence was observed mainly
along the notochord area (Figure 4).
Also it should be noted that when looking at the yolk of the eggs all of the maternal
GloFish eggs appeared to express the GFP (Figure 5) where none of the maternal
Zebrafish yolks expressed the GFP even though embryos later did express the
transgene.
Figure 3. Embryo of maternal mutant type GloFish and paternal wild type Zebrafish. GFP expression in significant amount of tissue observed.
Figure 4. Embryo of maternal wild type Zebrafish and paternal mutant type GloFish. GFP expression observed around notochord area.
Figure 5. Maternal GloFish yolk showing GFP expression before age where GFP should be expressed.
Myers 6
From our results we were able to conclude that the mode of transmission for
the transgene of GFP expression was autosomal dominant. This was determined
because if the transgene was autosomal recessive none of the embryos would show
a presence of GFP because none of the wild type would be heterozygous due to the
glow gene being transgenic in nature. But it was, however, able for the GloFish to be
both heterozygous as well as homozygous dominant. In our case of the maternal
GloFish it was almost certain that she was homozygous dominant for the transgene.
In the case of our paternal GloFish, in order to produce 4 out of 10 embryos with the
transgene he would have had to have been heterozygous for the transgene.
However, it also appears that there were some types of maternal effects at
play here. This is due mainly to two observations. The first observation being that
all of the eggs of the maternal Glofish contained yolks that appeared very light
green/yellow in regular light as well as fluoresced under blue light only 4 hours
after fertilization. None of the maternal Zebrafish yolks displayed this
characteristic. Secondly of all the embryos that showed GFP expression the maternal
Glofish embryos seemed to fluoresce in much more of their tissue than that of
maternal Zebrafish.
In order to fully examine this further, next time I would have more than just
one reciprocal cross. If the results showed the same maternal effects across several
matings we would definitely be able to conclude that not only autosomal dominance
is at play, but having a maternal Glofish also significantly alters how GFP is
expressed. Secondly, it would have been nice to let these embryos grow older to see
if the tissues in the 4 maternal Zebrafish embryos that expressed the transgene
Myers 7
eventually caught up with the other 39. Lastly looking at an F2 generation really
could have solidified what the mode of transmission is. However, for the amount of
time we had and the results we got, I feel confident in our autosomal dominant
conclusion.
Effort and Contribution
When dealing with live animals we had to keep them alive, keep water in the
tank and make sure the animals ate enough. As a group we worked well with
feeding them. There wasn’t a day when we crossed paths with each other and one
of our group members was checking with the other on feeding times and planning
on who was feeding throughout the week. Even though not everyone participated in
every event of the entire experiment I do feel like it was pretty evenly divided
amongst the group members and we each did a significant part, from keeping water
healthy, feeding, checking on fish, collecting eggs, examining and removing bad eggs,
setting up mating tanks, sexing the fish, taking pictures of eggs, and taking pictures
of a scoring fish. I did miss the scoring part, however, for everything else I was
present and put effort into keeping our fish alive, our embryos healthy and
preparing for mating.
Myers 8
Works Cited
GloFish® FAQ. (n.d.). Retrieved December 13, 2014, from http://www.glofish.com/about/faq/
Kimmel, C. B., Ballard, W. W., Kimmel, R. S., Ullmann, B., & Schilling, T. F. (1995). Stages of Embryonic Development of the Zebrafish. Developmental Dynamics, 203:253-‐310. Vick, B. M., Pollak, A., Welsh, C., & Liang, J. O. (2012). Learning the Scientific Method Using GloFish. Zebrafish, 9(4), 226-‐241. Doi:10.1089/zeb.2012.0758 Zebrafish FAQs. (n.d.). Retrieved December 13, 2014, from
http://www.neuro.uoregon.edu/k12/FAQs.html#high school
top related