Experiment 3 - Subcloning >> New Cell Line

What is the difference between cloning and subcloning?

Cloning is the process of isolating a piece of DNA and inserting it into a vector for replication

Subcloning is a specific type of cloning that moves an already cloned DNA fragment from one vector into a new or different vector

Goal of cloning

To capture and amplify a gene of interest for the first time

Goal of subcloning

Transfer an existing insert into a specialized vector

Ex. Moving from a basic cloning plasmid to an expression plasmid with specific promoters

Why would you need to perform PCR before doing a restriction digest?

1. To amplify a specific DNA sequence and engineer restriction sites onto its ends so it can be digested and cloned

2. To boost the DNA concentration for restriction digest, sometimes the target sequence is to physically sparse to manipulate so having billions of copies provides more material for the digest

What is the Flp-Inn T-REx system?

The system allows the generation of stable cell lines exhibiting tetracycline inducible expression of a gene of interest

3 major steps to create the TREX host cell line:

1. Integration of the plasmid containing the FRT site and the plasmid containing the tet repressor

2. Integration of an expression vector containing gene of interest under the control of the tet inducible promoter into the genome via Flp recombinase

What are the major components of the Flp-In T-REX system?

1. Flp - In target site vector

2. Tet repressor expression plasmid

3. Expression plasmid containing a FRT site

4. Flp recombinase expression plasmid

5. Control expression plasmid containing the CAT gene

2 plasmids that were transfected to make the host cell line (TREX cells)

1. pFRT/lacZeo target site vector

2. pcDNA 6/TR

What is the purpose of the pFRT/lacZeo target site vector?

• This vector contains the lacZ-Zeocin fusion gene. It’s expression is controlled by the SV40 promoter.

• It contains a FRT site which is the binding and cleavage site for the Flp recombinase.

• To identify a single integrated FRT site, after transfection cells were selected with Zeocin.

What is the purpose of the pcDNA 6/TR plasmid?

This plasmid expresses the Tet repressor under the control of the human CMV promoter

3 plasmids needed for the new TEL2 cell line

1. P0G44 Expression plasmid

2. PcDNA5/FRT/TO

3. Plasmid containing gene of interest

What is the P0G44 expression plasmid?

The plasmid containing Flp recombinase

AP258

What is the PcDNA5/FRT/TO plasmid?

The expression vector, the plasmid containing the gene of interest

AP257

What is the difference between a vector and a plasmid?

• vectors is a functional category

• plasmid is a description of a physical structure

A vector is a biological delivery vehicle, any DNA or RNA molecule used to carry foreign genetic material into another cell

A plasmid is circular double stranded DNA molecule that is physically separate from chromosomal DNA and can replicate independently, using coming from yeasts or e.coli

Types of vectors

1. Plasmids

2. Viral vectors (lenti, retro, AAV, adeno)

3. Cosmids

4. Bacteriophages

5. Artificial chromosomes

All plasmids are vectors

but not all vectors are plasmids

What kind of expression is the plasmid driving after the transfection?

Exogenous (introduced from the outside via foreign DNA)

What’s the big difference between expression during regular transfection and making the new cell line?

Transient = duration is limited to 24 - 96 hours

Stable cell line = permanent expression

Why is transient expression temporary?

DNA is diluted out and degraded as cells divide

Why is the expression in a stable cell line permanent?

The gene is passed down to all daughter cells indefinitely

What’s the main difference in what happens to DNA in a transient transfection vs using a stable cell line?

Transient = The plasmid remains in the nucleus, unintegrated (episomal)

Stable cell line = Plasmid DNA permanently integrates into the host genome

What’s the cell population like in a transient transfection?

Heterogeneous, the transfection efficiency varies

Some cells get 0 plasmids while others get hundreds

What’s the cell population like using a stable cell line for transfection?

Homogenous

Every single cell in the population carries the transgene

What’s a good reason to use a stable cell line instead of just transfecting?

Transient transfections can dilute the signal later shown on a WB because the untransfected cells can mask the phenotype of the transfected ones.

In a stable cell line, 100% of the cells carry the transgene

What do lab plasmids include? Key ingredients

1. Origin of replication (ori)

2. Multiple cloning site (MCs)

3. Selectable marker (antibiotic resistance gene)

4. Promoter