DNA delivery by microinjection for the generation of recombinant mammalian cell lines.
Gene transfer methods for producing recombinant cell lines are often not very efficient. One reason is that the recombinant DNA is delivered into the cell cytoplasm and only a small fraction reaches the nucleus. This chapter describes a method for microinjecting DNA directly into the nucleus. Direct injection has several advantages including the ability to deliver a defined copy number into the nucleus, the avoidance of DNAses that are present in the cell cytoplasm, and the lack of a need for extensive subcloning to find the recombinant cells. The procedure is described for two cell lines, CHO DG44 and BHK-21, using green fluorescent protein as a reporter gene. However, this method could easily be adapted to other cells lines and using other recombinant genes.
Chenuet
Sebastien
S
Ecole Polytechnique Féderale de Lausanne, EPFL-SV-IBI-LBTC, Lausanne, Switzerland.
Derouazi
Madiha
M
Hacker
David
D
Wurm
Florian
F
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004274:DNA, Recombinant / Q000378:metabolism*; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008322:Mammals / Q000378:metabolism*; D008845:Microinjections / Q000379:methods*; D010957:Plasmids / Q000302:isolation & purification; D011312:Pressure
D004274:DNA, Recombinant; D049452:Green Fluorescent Proteins