Name: reporter genes in cells and plasmids
Uploaded: 2021-04-07T12:12:00.000Z
Duration: 2 min 11 s
Channel: JoVE

K12 Standards

Tennessee Academic Standards

High School

Biology

links-among-engineering-technology-science-and-society

Links Among Engineering, Technology, Science, and Society

BIO1.ETS2.1

Reporter Genes

يستخدم الباحثون جينات المراسل لتحديد متى وأين يتم التعبير عن الجين ذي الأهمية. يرمز جين المراسل لبروتين يمكن تتبعه ، مثل البروتين ذو النشاط الأنزيمي المعروف أو البروتين الفلوري.
يتم التحكم في التعبير الجيني عن طريق التسلسلات التنظيمية لرابطة الدول المستقلة الموجودة في أعلى أو مجرى منطقة ترميز الجين في الحمض النووي.
يمكن تحديد نمط وتوقيت التعبير الجيني عن طريق إنشاء الحمض النووي المؤتلف مع جين مراسل تحت سيطرة تسلسل تنظيمي لرابطة الدول المستقلة ذي أهمية وإدخاله في الخلايا أو الكائن الحي.
يتم إدخال هذا الجين المؤتلف في أنواع مختلفة من الخلايا ، ويسمح لكل من الجين المعني وجين المراسل بالتعبير.
نظرا لأن الجين المعني والجين المراسل لهما نفس التسلسل التنظيمي لرابطة الدول المستقلة ، يتم التعبير عنهما في نفس الخلايا وفي نفس الوقت. لذلك ، من خلال مراقبة التعبير عن جين المراسل ، يمكن للعالم تتبع مكان التعبير عن الجين محل الاهتمام.
أحد الجينات المعروفة للمراسلين هو الذي يشفر بروتين يسمى بروتين الفلورسنت الأخضر - أو GFP.
تم اكتشاف GFP في قنديل البحر ، Aequorea victoria. ينتج البروتين مضان أخضر تحت الأشعة فوق البنفسجية ، مما يمكن الباحثين من تتبع موقعه داخل الخلية.
على سبيل المثال ، لدراسة التعبير عن β-tubulin في C. ايليجانس، تم استبدال تسلسل الترميز لجين β-tubulin بجين gfp.
أصبح التعبير الجيني gfp الآن تحت سيطرة المحفز لجين β-tubulin.
تم إدخال هذا الحمض النووي المؤتلف في الدودة باستخدام الحقن المجهري ، وتم التعبير عن الجين ، على غرار β-توبولين.
تمت مراقبة موقع GFP باستخدام الفحص المجهري الفلوري. نظرا لإظهار مضان GFP في الخلايا العصبية لمستقبلات اللمس ، فمن المحتمل أن يكون هذا هو الموقع الذي يتم فيه التعبير عن β توبولين.

<ol>
	<li>سوبوليسكي ، مارك ر. ، جيسون أوكس ، وويليام ب. هالفورد. "بروتين الفلورسنت الأخضر هو مراسل كمي للتعبير الجيني في الخلايا حقيقية النواة الفردية." مجلة FASEB 19 ، العدد 3 (2005): 1-20.</li>
	<li>بروغان ، جون ، فانغ لي ، وينرونج لي ، زيمين هي ، تشيان هوانغ ، وتشوان يوان لي. "تصوير المسارات الجزيئية: جينات المراسل". أبحاث الإشعاع 177 ، العدد 4 (2012): 508-513.</li>
	<li>سيرجانوفا ، إينا ، فلاديمير بونوماريف ، ورونالد بلاسبرغ. "جينات المراسل البشري: الاستخدام المحتمل في الدراسات السريرية". الطب النووي وعلم الأحياء 34 ، العدد 7 (2007): 791-807.</li>
</ol>

الجين المُخبِر

<div style="direction: rtl;">
جينات المرشدة هي نوع من جينات ترميز البروتين والتي غالبًا ما يتم تمييزها بالجين محل الاهتمام. بمجرد دخول الجينات المراسلة إلى الخلية المستهدفة، عادةً ما تنتج خصائص يمكن التعرف عليها بصريًا مثل التألق والتألق عند التعبير عنها جنبًا إلى جنب مع الجين محل الاهتمام. وبالتالي، فإن الجينات المراسلة "تبلغ" عن وجود أو عدم وجود الجينات ذات الأهمية في الكائن الحي، أو تحديد نمط التعبير الجيني، أو تتبع الموقع الفعلي لقطعة الحمض النووي أو البروتين في الخلية.

جينات المرشدة شائعة الاستخدام هي - GFP (جين البروتين الفلوري الأخضر)، lacZ (جين β- galactosidase)، RFP (جين البروتين الفلوري الأحمر)، وLuc (جين luciferase). تتوهج الخلايا التي تعبر عن GFP باللون الأخضر عندما تكون متحمسة بأطوال موجية من اللون الأزرق إلى نطاق الأشعة فوق البنفسجية، بينما تتوهج الخلايا التي تعبر عن RFP باللون الأحمر عندما تكون متحمسة بأطوال موجية تبلغ 488 نانومتر أو 532 نانومتر. تنتج الخلايا التي تعبر عن جين لوك إنزيمات لوسيفيراز التي تحفز التفاعل مع لوسيفيرين لإنتاج الضوء. يعد جين lacZ هو جين المراسل الأكثر شيوعًا المستخدم في الإشريكية القولونية. وينتج إنزيم β-galactosidase الذي يتسبب في ظهور البكتيريا باللون الأزرق عندما تنمو في وسط يحتوي على ركيزة X-gal.

الجينات المرشدة لدراسة نشاط المروج

إلى جانب استخدام الجينات المرشدة في دراسة التعبير الجيني والتوطين، فإنها يمكن أن تساعد أيضًا في دراسة قوة المروجين. يتم ذلك عن طريق وضع جين مراسل أسفل المحفز في البلازميد ثم إدخاله في خلية الثدييات. يراقب التعبير عن الجين المرشد نشاط المروج. إذا كان التعبير الجيني للمراسل مرتفعًا، يُقال إن المروج هو مروج قوي؛ إنه ضعيف إذا كان التعبير الجيني للمراسل منخفضًا.

يساعد الجين المرشد، عند وضعه أسفل المروج في البلازميد، أيضًا في قياس كفاءة النقل. يتم نقل البلازميد المؤتلف إلى خلية ثديية، ويظهر تعبيره أن الخلية قد تم نقلها بنجاح. تظهر كمية البروتين التي ينتجها البلازميد المؤتلف كفاءة نقل العدوي.
</div>

جينات المراسل هي نوع من جينات ترميز البروتين التي غالبا ما يتم تمييزها بجين ذي أهمية. بمجرد دخولها إلى الخلية المستهدفة ، عادة ما تكون جينات المراسل ...

Education

JoVE Core

Molecular Biology

البيولوجيا الجزيئية

Unit 2

الوحدة الثانية

Analyzing Gene Expression and Function

تحليل التعبير الجيني ووظيفته

KEY TERMS AND CONCEPTS

المصطلحات والمفاهيم الأساسية

reporter genes in cells and plasmids

Reporter genes are protein-coding genes used to track gene activity in cells. They are often tagged to a gene of interest so scientists can see whether that gene is present or active. When the tagged genes are expressed together, reporter genes can produce visible signals such as fluorescence or luminescence.

These signals help show the presence or absence of a gene of interest in an organism. They can also reveal the pattern of gene expression or the location of a DNA segment or protein inside a cell. Common reporter genes include GFP, lacZ, RFP, and Luc.

GFP stands for green fluorescent protein gene. Cells that express GFP glow green when exposed to blue to ultraviolet light. RFP stands for red fluorescent protein gene, and cells that express it glow red when excited by 488 nm or 532 nm light. The Luc gene produces luciferase enzymes, which react with luciferin to make light. The lacZ gene is the most common reporter gene used in E. coli. It produces β-galactosidase, which makes bacteria appear blue in media that contains X-gal substrate.

Reporter genes are also useful for studying promoter activity. A promoter is a DNA region that controls when a gene is turned on. Scientists place a reporter gene downstream of the promoter in a plasmid and then introduce the plasmid into a mammalian cell. The amount of reporter gene expression shows how active the promoter is. High expression suggests a strong promoter, while low expression suggests a weak promoter. Reporter genes can also help measure transfection efficiency, which shows whether the recombinant plasmid entered the mammalian cell successfully and how much protein it produced.

Reporter Genes in Cells and Plasmids

in vitro mutagenesis

In Vitro Mutagenesis for Gene Analysis

In vitro mutagenesis is a lab method used to change DNA and study gene function. It helps scientists test how specific DNA changes affect a gene’s activity and the traits it controls. A common approach is to make a known change in a DNA sequence and then examine the result. This can be done by deleting, inserting, or altering selected bases in a gene. The method is useful for linking a DNA change to a biological effect. Researchers use in vitro mutagenesis in molecular biology, genetics, and...

genetic screens

Using Genetic Screens to Find Disease Genes

Genetic screens are tools used to find genes and mutations linked to specific traits. They are also used to identify people or groups at risk for genetic diseases. That can support early intervention, targeted therapy, and reproductive choices. Forward genetic screens, also called classical screens, start with random DNA changes in an organism. Researchers may use radiation, mutagens, or added bases to create mutations that lead to visible phenotype changes. The mutant is then inbred so...

test cross

Test Cross in Genetic Inheritance

A test cross is a genetics tool used to determine the genotype of an organism with a dominant trait. It helps reveal whether the dominant individual is homozygous or heterozygous by crossing it with a recessive individual. The outcome of the cross shows the hidden genotype. If any offspring show the recessive trait, the dominant parent must carry one recessive allele. If all offspring show the dominant trait, the dominant parent is likely homozygous dominant. Test crosses are common in basic...

complementation tests

Using Crosses to Group Mutations

A complementation test uses a simple cross to find out whether two mutations are in the same gene or in different genes. It helps show how mutations are arranged on chromosomes. Edward Lewis first performed this test in the 1940s while studying fruit flies. To do the test, organisms heterozygous for different mutations are crossed pairwise in all combinations. If the mutations are in different genes, they can complement each other. In that case, each mutation provides the missing genetic...

single nucleotide polymorphisms-snps

SNPs as Genetic Markers and Variants

Single nucleotide polymorphisms, or SNPs, are small DNA changes at one specific genomic position. They are the most common type of sequence variation in the human genome. A point mutation counts as an SNP when it appears in more than 1% of the population.

bacterial transformation

Bacterial Transformation in the Lab

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transgenic organisms

Making Transgenic Organisms

Transgenic organisms are living things with DNA added from another organism. A transgene is the inserted gene. Scientists use this method to change an organism’s traits and to study how genes work. To make a transgenic organism, researchers first identify the gene they want to add. They then place that gene into the target organism’s DNA. Once the gene is integrated, the organism can pass it to new cells as it grows. This approach is useful in biology because it helps scientists analyze gene...

reproductive cloning

Cloning an Organism by Reproduction

Reproductive cloning creates a new organism that is genetically the same as the donor. It is different from cloning genes or cells because the goal is to make a whole living individual. This process usually relies on somatic cell nuclear transfer. In this method, the nucleus from a body cell is placed into an egg cell that has had its own nucleus removed. The egg can then develop into an embryo with DNA from the donor cell. Reproductive cloning is used to produce a copy of an organism, such...

crispr

CRISPR Gene Editing Basics

CRISPR is a gene editing tool that helps scientists make targeted changes to DNA. It uses a guide system to find a chosen sequence and a cutting protein to act at that spot. This method is based on a natural defense system found in bacteria. In high school biology, it is often studied as a way to see how genes can be edited with precision. CRISPR is widely used in molecular biology research. It helps scientists explore how genes function and how DNA changes can affect living things.

experimental rnai

RNAi for Gene Silencing and Discovery

RNA interference (RNAi) is a gene-silencing process used to reduce gene expression. It can block transcription or trigger RNA degradation. Scientists first discovered this mechanism in plants in 1998 through Andrew Fire and Craig Mello. RNAi has since been found in many eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. Researchers use it as a practical tool to study gene function. It can show what a gene does without permanently changing the organism’s DNA. <br...

reporter genes

Reporter genes are protein-coding genes used to track gene activity in cells. They are often tagged to a gene of interest so scientists can see whether that gene is present or active. When the tagged genes are expressed together, reporter genes can produce visible signals such as fluorescence or luminescence. These signals help show the presence or absence of a gene of interest in an organism. They can also reveal the pattern of gene expression or the location of a DNA segment or protein...

in-situ hybridization

ISH Probes for DNA and RNA Detection

In situ hybridization (ISH) uses labeled probes to detect and localize specific DNA or RNA molecules in cells, tissues, or tissue sections. The probe is a complementary strand of DNA or RNA that binds to a matching nucleotide sequence in the sample. Since its first use in 1969 for nucleic acid studies, ISH has become an important tool in research and in clinical diagnosis. Several probe types can be used in ISH. These include single-stranded DNA probes, double-stranded DNA probes, antisense...

chromatin immunoprecipitation- chip

ChIP Methods for DNA-Protein Binding

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synthetic biology

Synthetic Biology in Crops and Cells

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ribosome profiling

Ribosome Profiling for Translation Snapshot

Ribosome profiling is a deep sequencing method that gives a snapshot of active translation in a cell. It sequences the mRNAs protected by ribosomes. This shows which messages are being read to make proteins at a specific moment. The method has several uses in biology. It can track translation in a living organ or tissue type. It also helps measure new protein synthesis levels. Researchers use it to find translated open reading frames, which are stretches of RNA that can make protein. It can...

transgenic plants

Transgenic Plants for Better Crops

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gene therapy

Gene Therapy for Disease Treatment

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epistasis analysis

Gene Interaction Maps in Epistasis

Gene interaction maps in epistasis show how different genes work together to shape a phenotype. In epistasis, the interaction of genes or alleles at different locations affects the final trait. This often happens when one gene masks or interferes with the expression of another gene. Epistasis is common when genes are part of the same biochemical pathway. In that case, the expression of one gene can depend on a gene product made earlier in the pathway. Genes that mask or inhibit other genes in...

genome-wide association studies-gwas

Using SNP Patterns to Link Disease Risk

Genome-wide association studies, or GWAS, look for common SNPs, or single nucleotide polymorphisms, that may be linked to disease. Researchers compare how often a SNP appears in people with a disease and in people without it. When a SNP is seen more often in the disease group, it is considered associated with that disease. GWAS uses chi-square analysis to test the chance that an allele is linked to the disease. This approach does not require scientists to first find the target gene behind the...

key terms and concepts

Learn how reporter genes like GFP, lacZ, RFP, and Luc reveal gene expression, promoter strength, and transfection efficiency in cells and plasmids.

Reporter Genes in Cells and Plasmids | JoVE K12

molecular biology

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PCR - Polymerase Chain Reaction

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Complementary DNA

FISH - Fluorescent In-situ Hybridization

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Chromatin Immunoprecipitation- ChIP

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RNA-seq

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Transgenic Organisms

Gene Therapy

Reproductive Cloning

CRISPR

Experimental RNAi

RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. ...

Reporter genes are a type of&#160;protein-coding gene&#160;that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually ...

In-situ Hybridization

In situ hybridization (ISH) is a technique used to detect and localize specific DNA or RNA molecules in cells, tissue, or tissue sections using a labeled ...

Obtain, evaluate, and communicate information on how molecular biotechnology may be used in a variety of fields.
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Investigate means by which karyotypes are utilized in diagnostic medicine.
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BIO1.ETS2.2

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BIO1.ETS2.3

Reporter Genes in Cells and Plasmids

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