本期內(nèi)容是 實(shí)驗(yàn)十一：基因調(diào)控，實(shí)驗(yàn)?zāi)M請(qǐng)看下一期。本部分內(nèi)容來自 University of California, Berkeley - UC Berkeley Extension, 虛擬實(shí)驗(yàn)的內(nèi)容來自 Labster. 本部分內(nèi)容均不會(huì)標(biāo)記為為原創(chuàng)，但由于是UP主購買的課程，因此不接受非授權(quán)的轉(zhuǎn)載，謝謝您的理解。

每一個(gè)生物基礎(chǔ)實(shí)驗(yàn)均會(huì)分為三部分：第一部分為實(shí)驗(yàn)的生物理論；第二部分為實(shí)驗(yàn)的指導(dǎo)手冊(cè)；第三部分為 Labster 的虛擬實(shí)驗(yàn)?zāi)M。第一部分的基本信息由 Ying Liu, Ph.D. 提供，第二部分的實(shí)驗(yàn)手冊(cè)來自 Labster, 第三部分的實(shí)驗(yàn)?zāi)M過程由UP主操作。

Virtual Lab Manual 11:?Gene Regulation

Synopsis

Welcome to the future of medicine! In this simulation, you will learn the gene regulation principle that won the Nobel Prize for Physiology 2012. With the gene regulation technique developed?by Sir Gurdon and Shinya Yamanaka, your mission is to try to treat Amy, who is visually impaired, to try to help her regain her sight.

Learn the basics of stem cells

In order to help treat Amy, you will prepare induced pluripotent stem cells (iPS) from a fibroblast cells sample taken from the girl. The iPS cells will then be differentiated to retinal-pigmented epithelial cells for transplantation. In this simulation, 3D-animations will allow you to take a closer look at the machinery involved in gene regulation. You will learn about the basics of stem cells and what it takes to create one.

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Transform fibroblast cells into iPS cells

You will have to determine which transcription factors are essential in maintaining a stem cell’s features based on what you have learned in the previous mission. After determining the genes, you will use a retroviral infection technique to transform mature fibroblast into iPS cells. After incubation, you will examine which set of transcription factors is capable of inducing the fibroblast into stem cells (iPS).

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Analyze gene expression

By now you will have an idea of which transcription factor is capable of transforming fibroblast cells into iPS cells. The next step is to confirm your findings by Analyzing the gene expression in mRNA and protein level. You will perform reverse transcription PCR (RT-PCR) and Western blot to investigate the mRNA and protein level, respectively. Finally, you will have to decide which set of transcription factors can be used to transform fibroblast cells into iPS effectively. Will you be able?to help the doctor in treating Amy?

Learning Objectives

At the end of this simulation, you will be able to…

●?Explain how gene expression can be regulated

●?Describe the different levels of gene regulation (mRNA and protein)

●?Measure mRNA levels (RT-PCR), protein expression (Western blotting)

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Techniques in Lab

●?Microscopy

●?RT-PCR

●?Western blot

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Theory

Stem Cells

A stem cell is a single cell that can replicate itself and differentiate into other cell types. Stem cells do not have any tissue-specific structures that allow them to perform specialized functions. They are divided into groups depending on their differentiation abilities (potency).

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Gene Regulation

Gene regulation is important in all organisms. Both prokaryotic and eukaryotic organisms constantly turn on and off their genes in response to internal and external environmental conditions.

Early gene regulation conserves more energy compared to regulation in later stages. For example, selective blocking of transcription is much more energy efficient than waiting for the transcription and translation process to finish before finally degrading or inhibiting the protein. In prokaryotic organisms, gene expression is generally regulated during the transcription stage using operons. An operon is a cluster of genes with a single promoter that can be regulated by positive and negative controls.

Eukaryotic gene regulation

Most eukaryotic organisms are multicellular and are comprised of a variety of cells with different functions, regardless of having the same genome. To create different cell types, eukaryotic organisms rely on gene regulation. Gene regulation is important for maintaining the proper specialized functions of each cell. In eukaryotic organisms, gene regulation can occur at different stages:

Lentivirus

Retroviruses are lipid-enveloped particles comprised of linear single-stranded RNA genomes of 7 to 11 kilo bases. The main features of retroviral vectors are reverse transcription of the viral RNA genome into DNA, and stable integration into the host DNA. Lentivirus is a genus of retroviruses. Example of lentivirus is human immunodeficiency virus (HIV).

At the end of all retroviral genomes there are two long terminal repeat (LTR) sequences. The LTR sequences become the border of the?gag,?pol?and?env?genes. Lentivirus also encodes?tat?and?rev. The function of each gene is as follows:

●?gag: structural protein

●?pol: nucleic acid polymerase/integrases

●?env: surface glycoprotein

●?tat: regulatory protein for gene expression

●?rev: regulatory protein accessory genes

Retrovirus are desirable gene delivery vehicles or?vectors.

Reverse Transcription PCR

Not all genes are constantly expressed in all cells. A cell must transcribe the DNA sequence into mRNA in order to produce the encoded protein. Therefore, the level of gene expression corresponds to the level of mRNA. Gene expression can be measured using a reverse transcription polymerase chain reaction (RT-PCR). RT-PCR is comprised of these steps:

●?Conversion of RNA into cDNA using reverse transcriptase

●?Amplification of cDNA using PCR

mRNA is more fragile than DNA and cannot be amplified by PCR. For that reason, mRNA is converted into complementary DNA (cDNA). cDNA is DNA that is synthesized from messenger RNA molecules. cDNA synthesis is catalyzed by an enzyme called reverse transcriptase, which uses RNA as a template for DNA synthesis. Reverse transcriptase was initially discovered and isolated from a retrovirus. These viruses contain an RNA genome; therefore the viruses need to produce a cDNA copy of their genome to be compatible with the host cell's molecular machinery.

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The RT-PCR reaction comprises the following steps:

●?An oligo(T) fragment is used as a primer in order to bind to the 3’ poly(A)tail of each mRNA.

●?Reverse transcriptase uses RNA as a template to synthesize cDNA strands.

●?The resulting RNA-cDNA hybrid is separated by increasing the temperature.

●?A gene-specific primer anneals to its complementary sequence.

●?DNA polymerase produces the complementary DNA strand, starting from the primer binding site.

●?The strands are separated by increasing the temperature and the PCR cycle is repeated.

At the end of the RT-PCR reaction, there will be billions of copies of the gene of interest if the gene was expressed. The amount of DNA copies can be visualized by?gel electrophoresis.

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Western Blot

Western blotting or Western blot is an important method in protein identification. A Western blot can detect one specific protein in a solution that contains numerous other proteins. The term Western blot specifically refers to the transfer of proteins and their detection using antibodies.

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