Making zebrafish glow
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Making Zebrafish Glow!. Varun Bhadha Ruth McLaughlin Summer Research Connection The Center of Excellence in Genomic Science 24 July 2009. Photo provided by Frederique Ruf. How can we study early development?. Why study early development?. Developmental diseases

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Making zebrafish glow

Making Zebrafish Glow!

Varun Bhadha

Ruth McLaughlin

Summer Research Connection

The Center of Excellence in Genomic Science

24 July 2009

Photo provided by

Frederique Ruf


How can we study early development

How can we study early development?

Why study early development?

  • Developmental diseases

  • Understanding growth and organogenesis

  • Interesting and exciting

  • Localized expression

  • Co-injecting marker

  • Sub-cellular anatomy


Making zebrafish glow

A Solution: Fluorescent Proteins

Green Fluorescent Protein

jellyfish


Research goals

Research Goals

Generate localized fluorescent markers that will be injected into zebrafish embryos to study embryonic development

  • Membrane localized protein, cherry-red

  • Nucleus localized protein, cerulean-blue

Zebrafish cell

nucleus


Making zebrafish glow

Zebrafish as a Model Organism

  • Housing and husbandry

  • * adult fish (3-4cm) can be housed at high density - reduces costs (c/f mice)

  • * new strains and crosses can be easily bred (paired matings and crosses)

  • Developmental biology

  • zebrafish are vertebrates and have a gene complement very similar to humans

  • gene sequence is mostly done

  • development is rapid - organogenesis is virtually complete within 3 days

  • each female can lay >200 eggs per week

  • Imaging

  • high-resolution imaging of RNA and protein expression in whole embryos is easy

  • eggs are transparent, so early developmental processes can be easily visualized

  • transgenic and mutant lines expressing fluorophores can be easily imaged


Making zebrafish glow

Fish Room


Making zebrafish glow

chorion

cells

yolk

  • 4 cell stage, ~1 hours post fertilization (hpf)

  • chorion, embryo and yolk are all transparent

  • cells of the embryo can easily be injected with dyes, gene-specific morpholinos (to inactivate genes), or visualised by microscopy.


Making zebrafish glow

  • 3 dpf

  • organogenesis is almost complete.

  • the mouth, eye, otic vesicle (ear primordia), gut, liver, somites (muscles) & tail can all be seen.

  • majority of the embryo is still optically transparent at this age.


Making zebrafish glow

  • Adult zebrafish (female) 3-4 mpf

  • No longer transparent.

  • Sexually mature


Making zebrafish glow

Preparation of Genetic Material

plasmids

Restriction digest

Gel extractions

Gel electrophoresis

ligation

Bacterial transformation

Isolate new plasmid and clone

transcribe

inject


Dna plasmids

DNA Plasmids

  • Vectors, or plasmids, are circular strands of DNA used by bacteria.

  • Insert small fragments of DNA into bacterial plasmids that code for the coloring of the nucleus (cerulean-blue) and the cell membrane (cherry-red).

  • “Cut” or digest our plasmids using restriction enzymes.


Making zebrafish glow

Preparation of Genetic Material

plasmids

Restriction digest

Gel extractions

Gel electrophoresis

ligation

Bacterial transformation

Isolate new plasmid and clone

transcribe

inject


Restriction digest

Restriction Digest

Restriction enzyme cutting sites


Making zebrafish glow

Preparation of Genetic Material

plasmids

Restriction digest

Gel extractions

Gel electrophoresis

ligation

Bacterial transformation

transcribe

Isolate new plasmid and clone

inject


Gel electrophoresis

Gel Electrophoresis

  • Separates DNA fragments

  • Uses an agarose gel and an electrical charge

-

+

Gel box


Gel electrophoresis cont

Gel Electrophoresis (cont.)

  • Similar to children in a forest with the incentive of a candy bar

  • Forest = gel

  • Candy bar = charge

  • Children = DNA


Making zebrafish glow

Preparation of Genetic Material

plasmids

Restriction digest

Gel extractions

Gel electrophoresis

ligation

Bacterial transformation

Isolate new plasmid

transcribe

inject


Gel extraction

Gel Extraction

  • The DNA we need is now inside the gel.

    To get it out:

  • Cut DNA from the gel with razor blades

  • Extract the DNA from the gel piece


Making zebrafish glow

Preparation of Genetic Material

plasmids

Restriction digest

Gel extractions

Gel electrophoresis

ligation

Bacterial transformation

Isolate new plasmid

transcribe

inject


Ligation and transformation

Ligation and Transformation

  • Take the extracted DNA and “paste” together to form the desired plasmid

  • Insert the DNA into competent bacterial cells (transformation)

  • Plate the bacteria


Making zebrafish glow

Preparation of Genetic Material

plasmids

Restriction digest

Gel extractions

Gel electrophoresis

ligation

Bacterial transformation

transcribe

Isolate new plasmid and clone

inject


Dna replication and extraction

DNA Replication and Extraction

  • Isolate and grow bacterial colonies

  • Extract the DNA from the grown bacteria

  • Analyze the data to choose which bacterial DNA to use for the rest of the process


Dna extraction data

DNA Extraction Data

Tube # - DNA Concentration (ng/uL)

1- 56.2

2- 195.6

3- 20.0

4- 189.7

5- 82.8

6- 125.4

7- 428.2

8- 139.1

9- 125.3

10- 154.0

11- 108.4

12- 119.6

13- 159.9

14- 141.9

15- 86.8

16- 59.8


Dna linearization

DNA Linearization

  • The plasmid needs to be turned into a linear strand for transcription


Making zebrafish glow

DNA Linearization (cont)

Not1

DNA Plasmid

Linearized DNA strand

Not1

Not1


Making zebrafish glow

Preparation of Genetic Material

plasmids

Restriction digest

Gel extractions

Gel electrophoresis

ligation

Bacterial transformation

transcribe

Isolate new plasmid and clone

inject


Dna strand to rna

DNA Strand to RNA

  • To inject into the embryo, we will need to use RNA

  • Therefore, we must transcribe RNA from the DNA.

https://eapbiofield.wikispaces.com/file/view/c7.17.7b.transcription.jpg


Making zebrafish glow

Preparation of Genetic Material

plasmids

Restriction digest

Gel extractions

Gel electrophoresis

ligation

Bacterial transformation

transcribe

Isolate new plasmid and clone

inject


Injecting

Injecting

~30 min pf

1 cell stage of embryo

Single cell

  • RNA is introduced into the cell by injection at the 1 cell stage.

Injection needle

Size - 0.7 mm

http://images.google.com/imgres?imgurl=http://www.biocompare.com/images/bc/006/ArticleImages/AAA_1.jpg&imgrefurl=http://www.biocompare.com/Articles/TechnicalArticle/1657/Microinjection-Of-DNA,-RNA-And-Tracer-Dyes-Into-Early-Fish-Embryos-from-Eppendorf-AG.html&usg=__mjTx6MdnzrmnbTeV0SbGSsp-E6U=&h=495&w=904&sz=301&hl=en&start=34&tbnid=JZiQGvLg5c2jrM:&tbnh=80&tbnw=147&prev=/images%3Fq%3Dzebrafish%2Binjections%26gbv%3D2%26ndsp%3D21%26hl%3Den%26sa%3DN%26start%3D21%26newwindow%3D1


Conclusions

Conclusions

  • Genetic material (DNA/RNA) can be manipulated ( as in removed, changed, and inserted) between species.

  • We have successfully manipulated zebrafish embryos to cause them to fluoresce


Acknowledgements

Acknowledgements

Thank you to…

Fraser Laboratory

Dr. Aidyl Gonzalez-Serricchio

Ilana Solomon

Dr. Scott Fraser

Kristina L. Hilands

Leigh Ann Fletcher

SRC Administrators

James Maloney

Dr. Sherry Tsai

Funding

Siemens Foundation

Howard Hughes Medical Institute

Pasadena Independent Schools Foundation

Caltech

Oak Crest Institute of Science

Our Families

Our fellow SRC researchers


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