Protein expression in the mammalian cells

In summary, it is recommended to add mRNA destabilization signals to the 3' end of the cDNA in order to reduce mRNA stability. The most commonly used homopolymeric tails for this purpose are polyA or polyT. Introns are necessary for gene expression in some cases, and should be added to the cDNA. Splicing sequences should also be included in the cDNA, either on the plasmid or on the insert, depending on the presence of introns. The recommended location for adding splicing sequences is close to the intron-exon boundaries.
  • #1
Goodie
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# Do I need mRNA destabilization signals for my gene expression? If so where should I add it?

# It says; "cDNA are often obtained by addition of homopolymeric tails into the 5’ non coding region which should be removed.” Which homopolymeric tails are usually used for this purpose?

# Which sequences on the 3’ end that reduce mRNA stability?

# It says that some genes need introns for expression hence I have to add it in my cDNA. How do introns look like and where should I add it?

# Should splicing sequences be already on the plasmid or should I add it on my insert? Should I add it only when I have introns on my insert or should i always have to add it? If so where should I add it on my insert? How do the splicing sequences be like?


Thanks for any inputs. o:)
 
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  • #2
In general, for recombinant protein expression in mammalian cells, you would clone only the protein-coding sequence of a gene into a plasmid. You would not clone in any of the 5' or 3' untranslated sequences from the cDNA nor would you include any introns.
 

1. How do you define protein expression in mammalian cells?

Protein expression in mammalian cells refers to the process by which genetic information is translated into functional proteins within the cells. This involves the transcription of DNA into mRNA and the subsequent translation of mRNA into amino acid sequences, which then fold into specific protein structures.

2. What are the key factors that affect protein expression in mammalian cells?

The key factors that affect protein expression in mammalian cells include the type of cell used, the type of vector or plasmid used for gene expression, the strength of the promoter used, the stability of the mRNA produced, and the efficiency of translation and post-translational modifications.

3. What are the common methods used for protein expression in mammalian cells?

Some common methods for protein expression in mammalian cells include transient transfection, stable transfection, viral transduction, and protein expression using cell-free systems. Each method has its own advantages and limitations, and the choice of method depends on the specific research needs.

4. How can protein expression in mammalian cells be optimized?

Protein expression in mammalian cells can be optimized by using appropriate promoters, optimizing the culture conditions, and optimizing the vector or plasmid design. Additionally, methods such as codon optimization, gene editing, and the use of fusion tags can also improve protein expression levels.

5. What are the applications of protein expression in mammalian cells?

Protein expression in mammalian cells has a wide range of applications in basic research, drug development, and biotechnology. It is used to study protein structure and function, to produce therapeutic proteins, and to generate animal models for disease research. It is also a crucial step in the production of biologics such as vaccines and antibodies.

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