Difference between revisions of "AGiR! for genomic integrity"

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(Main Goal)
(Objectives)
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=== Objectives ===
 
=== Objectives ===
* Demonstrate methods and Setup lab equipment for the cheek cell 'comet' and micronuclei assays
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* Obtain ready results with cheek cells for the comet and micronuclei assays
::* Microscope
+
* Put together the open source protocol, with suggestions for standard tests and controls
::* Mercury Lamp Setup and filters for fluorescent dye detection.
+
* Demonstrate methods and share the love with other biohacker groups (La Paillasse etc.)
::* Agarose, glass slides, and gel running apparatus for embedding cells, treatments and nucleoid migration (for the comet assay)
+
::* Other reagents and histochemical stains (triton, protease, EDTA, methylene blue, buffer solutions, etc.)
+
::* Hydrogen peroxide for positive control cells (oxidative damage)
+
::* Antioxidant molecules as possible 'protective' intervention
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::* Could also try to design a transilluminator suitable for cellular resolution (UV/Fluo as usual for comets) when imaging (to look for open source models)
+
  
 
Cheek cell epithelia are about 50 micrometers in diameter. [[File:Cells3.jpg|200px|thumb|right|Three cheek cells imaged on the scope at the Hackuarium. Some methylene blue stain may be visible in the most 'in focus' nucleus - left, middle cell. ]]
 
Cheek cell epithelia are about 50 micrometers in diameter. [[File:Cells3.jpg|200px|thumb|right|Three cheek cells imaged on the scope at the Hackuarium. Some methylene blue stain may be visible in the most 'in focus' nucleus - left, middle cell. ]]
  
  
* Study the feasability of an assay on either bacterial or bulk DNA
+
* Study the feasability of further methods that could contribute to this project - i.e. an assay on either bacterial or bulk DNA
  
 
== Background & Inspiration ==
 
== Background & Inspiration ==

Revision as of 06:37, 18 May 2016

MinilogoB.jpg

Genomic integrity is a new 'big picture' concept to aid public health efforts and basically includes all the molecular genetic details of cells! So many things we commonly choose to do can impact genomic integrity, also potentially affecting future generations!

At the Hackuarium, AGiR! hopes to really get some citizen science action going - developing DIYbio methods to readily assay *your very own* cells for DNA damage, using classic (circa 1980s) techniques. If these can still be sold in 'kits,' maybe it will also be simple to make them available to all for open source efforts?
Here is the basic plan: using inner cheek cells for micronuclei and comet assays!
Watch this space for more info and visit
the AGiR! site


Introduction

Context

This is a part of a new Hackuarium project on genomic integrity.
This wiki page is under construction, and will help keep track as we develop open source simple protocols of two classic methods for quantitation of DNA damage in human cells, the micronuclei and comet assays.

People

Contact and lead:

  • Rachel Aronoff

Participants:

  • maybe you?!

Main Goal

Genomic integrity is a big picture concept which includes basically all the molecular genetic details of cells (from RNAs to DNAs and the epigenetic marks for proper gene expression), and thus is a topic that can be explored in several ways. For the genomic integrity project at the Hackuarium the focus will be on direct DNA damage. Comet and micronuclei assays were developed in the 1980s and publications about use of these assays on human cheek cells are already in the literature. These methods, as open source protocols, will encourage citizen science and raise awareness. Additionally, they can provide an easily "workshopable" way to show people more about their own cells, while learning how to assess DNA damage.
In other words, this experiment can be used as a gateway, to demonstrate and raise awareness for everyone on the damage possible from many common substances or activities - on your own cells, your own genetic information!
In the scope of Hackuarium activities, mammalian cell cultures are still (april 2016) a no go, but freshly isolated cheek cells from individuals can be used to do tests on DNA damaging compounds or activities.
In order to further investigate compounds that might cause DNA damage, a classic test of bacterial reversion (i.e. the Ames test) or on bulk DNA may also be nice to establish at the Hackuarium.
If enough people follow the 'open source' protocol(s) that we develop to test substances on their cheek cells, and clearly significant effects are observed, we may even be able to publish such effects.

Objectives

  • Obtain ready results with cheek cells for the comet and micronuclei assays
  • Put together the open source protocol, with suggestions for standard tests and controls
  • Demonstrate methods and share the love with other biohacker groups (La Paillasse etc.)
Cheek cell epithelia are about 50 micrometers in diameter.
Three cheek cells imaged on the scope at the Hackuarium. Some methylene blue stain may be visible in the most 'in focus' nucleus - left, middle cell.


  • Study the feasability of further methods that could contribute to this project - i.e. an assay on either bacterial or bulk DNA

Background & Inspiration

Here is the link to the Prezi Rachel presented (13apr16) at an Open hackuarium night! Prezi on DIYbio for Genomic integrity!

Some theory

More on comet cells and micronuclei to come soon! But in the meantime, here are the references to the cheek cell papers mentioned in the presentation (and in the prezi):
Szeto et al (2005) Mutation Research 578:371-381
(In this paper, green tea is also shown to be protective.)
dos Santos Rocha et al (2014) Genetics and Molecular Biology, 37(4)702-707
(This paper looks at micronuclei in cheek cells (and also analyses apoptosis) from mouthwash and alcohol users.)

Inspiration / References / Similar projects

Inspiration: good pages to come

  • [http://]
  • [Link 2]


Setup description

General principle:

Materials & budget

  • What, size | quantity | where to get it | price / unit


(If it’s an experiment) Parameters

To get baselines first

then To vary | Values

p1 |
p2 |
Constants (Key elements of set-up)
c1 |
c2
To measure/ observe
m1
m2

Protocol (Plan)

Startup

Phase 1

do what | frequency | duration

Lab Notebook & News (Real)

Protocols and measurements to be linked from here: [link to other doc]

Run / prototype #2

What's new compared to #1:

Results / Feedback from use

Recommendation + next steps