Promocionado por PROMEGA NL
Universidad catholica de Louvain, Facultad de Medicina 


Atelier de genetica molecular SBIM 2520
expresion en mamiferos
monday 08 July, 2002
monday 05 August 2002
por Jean-Pierre HERVEG MD herveg@bian.ucl.ac.be.nospam cut .nospam
go back to
first week, program


1. plan de trabajo del dia

1. transforma la bacteria para obtener suficiente plasmido.
2. prepara la
ampicilina
3.
prepara las celulas de mamifero (HEK 293) que van a ser transinfectadas con este plasmido.

a. prepara las celulas HEK293.
b.
Cuenta las celulas HEK293.

2. Material de trabajo a utilizar hoy:

1.Camara de flujo laminar
2.
Estufa con O2/CO2
3. Erlenmeyer con
medios de cultivo
4. Sembrado y aislado de las celulas de mamifero

recuerda que este es un Atelier y no un curso solo teorico de ensenanza
...........................




3. Teoria y ejercicios

1. transfeccion de celulas de mamifero
2. prepara suficiente plasmido recombinante.
3. Aisla y cuenta las celulas de mamiferos.

..........................

1. transfection of mammalian cells

"Transform" a bacteria means to introduce a DNA molecule in this bacteria. When a DNA molecule is introduced in eukaryotic cell, the process is called transfection (transformation in eukaryotes has another meaning, it means cancerization).

You will introduce a recombinant plasmid in the mammalian cell HEK293. The plasmid foreign insert code for a gene that will be ewpressed in these HEK293 cell. Our plasmid foreign gene codes for ß-galactosidase, an enzyme, which splits lactose into galactose and glucose (the Cell by GM Cooper page 230-231). Regular plasmids can only express genes in prokaryotic cells. These prokaryotic genes are not recognized by the eukaryotic machinery.

......

To be recognized by the eukaryotic machinery, a gene should be theoreticaly monocystronic, have a eukaryotic promoter (CMV, SV40, etc.), an intron at its 5' end, and a poly(A) tail.

Transfection yields are very low. The few transfected cells have to be detected. Here transfected eukaryotic cell would express ß-galactosidase and show a ß-galactosidase activity. This activity will be recognized by a color reaction which will stain the cells in blue with specific reagent.

We will detect the cells which synthesize ß-galactosidase with a microscope, using a staining reaction.

 

REMEMBER THE lac operon (Dr Etienne DE PLAEN)


2. You should first prepare enough recombinant plasmid.

To achieve this first goal, you will need to Transform competent bacterial (JM109) cells by the recombinant plasmid. using the calcium chloride procedure. Multiplying the transformed bacteria will multiply the recombinant plasmid.

Describe a plasmid able to express an inserted gene in a mammalian cell, insisting on how it differs from the usual plasmids, which express their genes in bacteria. Watch and describe the plasmid below.

Start your description from the BlgII site, and scan the plasmid clockwise. You are supposed to be able to describe everything. Let's suppose now that you are in a lab and have to order this plasmid, what information should you need?

 

................

Let's suppose that our gene is inserted between the sites EcoRI and Not I. Try to order the two enzymes and their buffers. You'll learn to order yourself and discover how expensive they are.

3. prepare the mammalian cells (Isolate and count them).

We will transfect HEK-293 cells with the plasmid. HEK-293 cells are human embryonic kidney cells. They are routinely grown in our university. You will got them in a culture plastic flask containing an orange colored growth medium. The cells adhere to the bottom of the flask. They multiply, trying to cover the whole bottom. When the bottom is covered, cells are said to be confluent. Dead cells float on the surface of the medium.

1. You will have to observe the cells attached to the bottom of the flask. This observation requires a special microscope, an inverted microscope. This will tell you if the cells are confluent or not. Why we should use an inverted microscope, and not an ordinary microscope?

2. You will have to detach cells from the flask bottom, to suspend and to count them.

3. Finally you will have to grow them (2 million per box).

...................

Sponsored by PROMEGA NL
Université catholique de Louvain, Faculté de Médecine 


molecular genetics workshop SBIM 2520
mammalian expression
monday 08 July, 2002
by Jean-Pierre HERVEG MD herveg@bian.ucl.ac.be.nospam cut .nospam
go back to
first week, program