Morpholinos, Zebrafish, and Reverse Genetics By Seth Hamilton and Tim Rose

University of Minnesota Duluth: Genetics (BIOL 2202)

Background Information:

Reverse genetics: Determining a particular gene’s function by analyzing the effect on a phenotype after some form of genomic modification.

Other Current Techniques

1. RNAi 2. CRISPR-Cas9

3. TALENs

Table 1: Outcomes of Chordin Morpholino injections

14% of embryos that survived the Chordin MO injection displayed the mutant phenotype.

Some damage is caused during the invasive injection process, leading to embryonic death.

Mutant embryos displayed an engorged tail and shrunken head, in contrast to the wildtype embryos (Figure 1).

The un-injected control embryos all died. This is likely due to a programmed cell death pathway, in which the death of one embryo signals the death of the rest.

Chordin Morpholino: Decreases expression of chordin gene by blocking translation. Chordin Protein: Responsible for the morphogenesis of Danio rerio’s skeleton.

Hypothesis: Following Chordin MO injections, some embryos will

display the mutant phenotype, which is represented by stunted, skeletal morphogenesis.

Morpholino (MO): An oligomer molecule used to modify gene expression.

Mechanisms to Decrease Gene Expression

1: Normal Gene Expression DNA mRNA Protein 2: Morpholino Blocks Translation DNA mRNA No Protein 3: Morpholino Blocks Splicing DNA mRNA Modified Protein

Step 1: Chordin Morpholino Injections 53 students each injected the Chordin MO into the yolk of

about 4 newly-spawned embryos.

Step 2: Embryo Fixation Embryos were fixed in paraformaldehyde 24 hours

post-injection.

http://www.alsliga.be/sites/default/files

http://www.devbio.biology.gatech.edu/wp-content/uploads/2012/04/zf-300x225.jpg

Mutant WT

http://fr.academic.ru/pictures/frwiki/66/Blocking_Splicing.gif

http://www.practicalfishkeeping.co.uk/custom/images/medium/55684d1b13886.jpg

http://www.nature.com/nmeth/journal/v10/n4/images/nmeth.2415-F1.jpg

Pros Inexpensive Promoter Specific Controllable Degree of Knock-down

Cons Not All Organisms are Susceptible

No Knock-out

Pros Simple Target Design Very Efficient Multiple Mutations Can be Added at Once

Cons Nonspecific Binding Strong Mosaicism Pros Nucleotide Specific Relatively Easy to Design

Cons Nonspecific Binding Currently Limited to Model Organisms

http://schaechter.asmblog.org/.a/6a00d8341c5e1453ef01a511d0f3f1970c-300wi

Date # Embryos # Alive WT Mutant Other

10/15/2015 53 37 29 4 4

10/16/2015 52 27 23 3 1

11/14/2015 41 29 17 6 6

11/20/2015 88 20 17 3 0

Figure 1: Fixed embryos 24 hours after injection. Embryos on the left (A , C) were injected with the Morpholino and displayed the wildtype phenotype. Embryos on the right (B, D) were injected with the Morpholino and displayed the mutant phenotype.

Introduction and Methodology:

Discussion:

Results: Future of Reverse Genetics: