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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  

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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  

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  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  

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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.  

 

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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.  

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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  

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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.  

 

 

 

 

 

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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