Using Poly-A Sequences & Isogenic/Null Cell Lines for Reporter Genes

In summary, the poly-A sequence is typically found downstream of a gene to aid in RNA termination and translation during eukaryotic DNA transcription. However, in certain cases, it may be found upstream of a reporter gene to protect the fused protein. Isogenic cell lines have a similar genome and null cell lines lack a specific trait.
  • #1
Goodie
20
0
• Why should some Poly-A sequences lay upstream of a reporter gene?
• Isogenic cell lines?
• Null cell lines?

Any ideas are greatly appreciated.
 
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  • #2
Goodie said:
• Why should some Poly-A sequences lay upstream of a reporter gene?

You should find the answere if ask yourself: What is the role of the polyA sequence/tail during eukaryote DNA transcription?


Goodie said:
• Isogenic cell lines?

Isogenic means similar genome/genotype.

Goodie said:
• Null cell lines?

A null mutant usually lack a specific trait.
 
  • #3
iansmith said:
You should find the answere if ask yourself: What is the role of the polyA sequence/tail during eukaryote DNA transcription?

Usually the poly A sequence lays downstream of a gene to give it good RNA termination and translation. BUt when it lies upstream is something new :bugeye: . Upstream means before the promoter. How come?
 
  • #4
Is the polyA upstream of the promoter for the reporter gene (lacZ) or upstream of selection marker (noemycin resistance) promoter.

The reporter gene will be inserted in the gemone and will cut the gene X and the normal polyA will not protetect the fused protein. The poly-A upstream of the selection marker promoter is there to protect the fused protein (geneX-LacZ). The neomycin resistant marker will fused with the rest of the protein and the mRNA will be protect by the geneX polyA tail.
 
  • #5
iansmith said:
Is the polyA upstream of the promoter for the reporter gene (lacZ) or upstream of selection marker (noemycin resistance) promoter.
The polyA is upstream of the promoter for the reporter gene

iansmith said:
The reporter gene will be inserted in the gemone and will cut the gene X and the normal polyA will not protetect the fused protein.
Can you explain more of this?

iansmith said:
The poly-A upstream of the selection marker promoter is there to protect the fused protein (geneX-LacZ). The neomycin resistant marker will fused with the rest of the protein and the mRNA will be protect by the geneX polyA tail.
Where did you get this?

Thanks.
 
  • #6
Look at figures 1 and 2 on this page. It will explain some concept.

http://faculty.virginia.edu/mammgenetics/805-3rd03.html
 
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Related to Using Poly-A Sequences & Isogenic/Null Cell Lines for Reporter Genes

1. What are poly-A sequences and how are they used in reporter genes?

Poly-A sequences are long stretches of adenine nucleotides found at the end of eukaryotic mRNA molecules. They are important for stabilizing the mRNA and are required for efficient translation. In reporter genes, poly-A sequences are often added to the end of the gene of interest to increase its stability and expression levels.

2. What is the difference between isogenic and null cell lines?

Isogenic cell lines are genetically identical and differ only in the gene of interest, while null cell lines have a specific gene or genes intentionally removed or disrupted. Isogenic cell lines are useful for studying the specific effects of a gene, while null cell lines can be used to determine the function of a particular gene.

3. How are poly-A sequences and isogenic/null cell lines combined in reporter genes?

Poly-A sequences can be added to the end of a gene using molecular cloning techniques. Isogenic or null cell lines can then be transfected with the reporter gene construct using methods such as electroporation or viral transduction.

4. What are the advantages of using poly-A sequences and isogenic/null cell lines in reporter genes?

Poly-A sequences increase the stability and expression levels of the reporter gene, making it easier to detect and study. Isogenic and null cell lines provide a controlled genetic background, allowing for more accurate and specific analysis of the gene of interest.

5. Are there any limitations or considerations when using poly-A sequences and isogenic/null cell lines for reporter genes?

Poly-A sequences may not be suitable for all genes, and their addition may alter the function of the gene of interest. Isogenic and null cell lines may not fully reflect the complexity of gene interactions in a whole organism. Additionally, careful controls must be used to ensure that any observed effects are due to the reporter gene and not other factors.

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