title: DNA delivery by microinjection for the generation of recombinant mammalian cell lines.
abstract: 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.
authors.affiliation: Ecole Polytechnique Féderale de Lausanne, EPFL-SV-IBI-LBTC, Lausanne, Switzerland.
authors.forename: Sebastien
authors.initials: S
authors.lastname: Chenuet
authors.forename: Madiha
authors.initials: M
authors.lastname: Derouazi
authors.forename: David
authors.initials: D
authors.lastname: Hacker
authors.forename: Florian
authors.initials: F
authors.lastname: Wurm
mesh_terms: 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
chemical_list: D004274:DNA, Recombinant; D049452:Green Fluorescent Proteins