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An Introduction to Some Molecular Biological Techniques Used in Neuroscience

Objective 6: Cite some examples of the use of molecular biological techniques in neuroscience.

Objective 6 Video Lecture

 

In many cases, neuroscientists would like to measure the amount of mRNA transcript which is being made in a particular cell or tissue at a particular time.

 

Diagram showing transcriptional control of genes involved in zebrafish models of autism.
An example of how neuroscientists use differences in gene expression to better understand how the brain works. This example is from a zebrafish model of human autism. NPC, neural progenitor cell. From https://www.frontiersin.org/journals/molecular-neuroscience/articles/10.3389/fnmol.2020.575575/full

For example, we might want to know what genes are “turned on” (seen as a rise in specific mRNA levels) during cell migration. These investigators have found that methyl CpG binding protein 2 (MECP2) is increased in neuronal progenitors that are in the process of migration. (If you want to investigate any of the other genes listed, use your favorite search engine with the gene name plus “GeneCards” — e.g.

MECP2 GeneCards

— or simply search inside the GeneCards site.)

Diagram showing the steps of reverse transcriptase-polymerase chain reaction.In the reverse transcriptase – polymerase chain reaction method, we carry out the following steps in order:

  • total RNA is isolated
  • the RNA which has poly(A) at the 3′ end is isolated from total RNA (this is presumed to be mRNA, since only mRNA carries a poly(A) tail
  • the enzyme reverse transcriptase converts RNA to DNA, now called complementary DNA (cDNA)

Thermostable DNA Polymerase

Before the discovery of thermostable DNA polymerases, it was theoretically possible to carry out a polymerase chain reaction experiment, but the investigator had to add new DNA polymerase each cycle to replace that destroyed by the 95°C heating step. That was obviously tedious.

Thermus aquaticus (shortened to Taq) was first discovered by Brock and Freeze (!) in Mushroom Spring in the Lower Geyser Basin of Yellowstone National Park. T. aquaticus grows best at 70-75°C, a temperature that will burn human skin. Seven years later, Alice Chien and colleagues isolated a thermostable DNA polymerase (called Taq polymerase) from this organism. The discovery of this bacterium, and similar thermophiles (“heat-lovers”), has revolutionized molecular biology.

 

 

 

 

In the polymerase chain reaction:

  • the investigator has made two primers with an included fluorescent tracer (F), about 100 or so bases apart, which are complementary to opposite strands of DNA
  • the DNA is denatured by heating (typically to 95°C)
  • the primers are annealed to both strands of the DNA (typically at about 50°C)
  • a thermostable DNA polymerase binds to the double-stranded region (where the primer is annealed) and extends the double-stranded sequence (typically at about 72°C)

 

 

 

Diagram showing how quantitative RT-PCR is used to detect the level of specific mRNAs in neural tissue.With each annealing cycle, the primers bind to each of the two strands of the double helix, facing off about 100 base pairs apart. Then, during the 72°C extension cycle, the thermostable DNA polymerase builds a new strand starting at the primers, each going 5′ to 3′ but in opposite directions. This builds two new strands with each cycle, which can then anneal to form twice as much double-stranded DNA as before the start of the cycle. One double-stranded DNA molecule becomes two, then four, then eight, and so forth. By the end of 30 cycles, each cDNA molecule is now 1,073,741,824 (230) DNA molecules. You can see that even a small handful of cDNA molecules in the original sample will be detectable after this many cycles. The cycle number where the threshold of detectability is crossed (Ct) depends on the concentration of cDNA in the original sample. A low Ct indicates a high initial concentration of that particular cDNA, while a high Ct indicates a low initial concentration. In this way, the amount of mRNA in the original sample can be determined relative to the concentration of a “reporter” or “housekeeping” gene.

One significant limitation of RT-qPCR is that the investigator can only amplify one gene at a time. There is a need, in many experimental systems, to examine the amount of dozens or hundreds of mRNA species simultaneously. For example, as can be seen above, multiple genes interact in synaptic development. Many times one starts an experiment with no idea what those genes are. Or, in this example, a pathogen damages a cell, and we need to know which genes have been up-regulated, which down-regulated, and which are unchanged.

Diagram showing how gene arrays are used to measure gene expression.To help investigators understand what is happening in these circumstances, cDNA microarrays (gene arrays) were invented. This allows hundreds of genes (typically, 640) to be assayed simultaneously and compared between two different tissues — for example, two different developmental stages, such as before and after synaptogenesis, or two different states of health, such as normal and cancerous.

Diagram showing how RNA sequencing for transcriptome analysis is performed.DNA microarrays have fallen out of favor and a much more cost-effective and accurate way to get the same information has been invented: RNA sequencing.

Infographic showing how transgenic mouse lines are created.The 2007 Nobel Prize in Physiology or Medicine was awarded to Mario Capecchi, Martin Evans, and Oliver Smithies for the technique that came to be known as development of transgenic mice. Evans discovered the embryonic stem (ES) cell of the mouse embryo. This is the one cell in the early embryo that becomes 3,000,000,000 adult mouse cells or 10,000,000,000 adult human cells.

Novel DNA is introduced into ES cells by a technique called electroporation, and the modified ES cell can then be injected into a mouse embryo and implanted into a foster mother. The modified gene is now stable and can be transmitted to offspring in the usual way.

Two kinds of transgenic modifications are possible: knockout, where a normal gene is removed; and knock-in, where a novel gene is added.

Infographic showing the creation of conditional knockout mice.Often, knocking a gene in or out results in a dead animal. To avoid this, or to refine the location of the modified genes, one can build a tissue-specific promoter into the mouse genome just ahead of the knockout (or knock-in) gene.

Infographic showing applications of CRISPR technology.An even more targeted technique is based on a primitive defensive system used by bacteria, the CRISPR/Cas9 system.

 

Diagram showing the operation of a CRISPR system.

The gene sequence that is used to locate the cut is called a Clustered Regularly Interspaced Short Palindromic Repeat, or CRISPR, and the protein that does the cutting is called Cas9. The CRISPR/Cas9 system can be used to accurately edit specific gene sequences to whatever configuration the researcher wants.

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Introduction to Neuroscience Copyright © by Jim Hutchins; Lindsey Aune; and Rachel Jessop is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License, except where otherwise noted.