TY - JOUR
T1 - Targeted Inactivation of the G12α gene with replacement and insertion vectors
T2 - Analysis in a 96-well plate format
AU - Rudolph, U. w.e.
AU - Bradley, Allan
AU - Birnbaumer, Lutz
N1 - Funding Information:
Research was supported by the Howard Hughes Medical Institute and the American Heart Association (Clinician Scientist Award to R.M.M.).
Funding Information:
We are especially indebted to Dr. Paul Hasty, and members of the Bradley laboratory for sharing expertise. We thank Dr. Philippe Soriano for pNeo with the PolII-Neo-bpA cassette, Dr. Paul Hasty for the pKS(Xho)MCITK cassette, and Dr. David Nelson for the BALB/c-derived ~.EMBL3 library. U.R. was supported by a fellowship from the Deutsche Forschungsgemeinschaft. L.B. is supported by grants from the National Institutes of Health. A.B. is an Associate Investigator of the Howard Hughes Medical Institute.
PY - 1994/1/1
Y1 - 1994/1/1
N2 - This chapter investigates the targeted inactivation of the Gi2α gene with replacement and insertion vectors. It has become possible to inactivate genes in mammalian cells by introducing a mutation at a specific genomic locus. Murine embryonic stem cells (ES cells) have been isolated that can be genetically manipulated in cell culture and reintroduced into blastocysts, giving rise to chimeric animals. Chimeras may transmit the mutation to their offspring, which will then be heterozygous for the mutation. The mutation can then be bred to homozygosity, enabling the analysis of the mutational phenotype both at the level of the whole animal and at the cellular and molecular level by analyzing cell lines or tissues derived from animals homozygous for the desired mutation. An important task in gene targeting experiments is to identify the rare targeted events over the background of random integrations. Most protocols include the use of a Neo cassette in the targeting vector disrupting an exon of the gene of interest and enabling a positive selection for cells that have stably integrated the transfected DNA into the genome.
AB - This chapter investigates the targeted inactivation of the Gi2α gene with replacement and insertion vectors. It has become possible to inactivate genes in mammalian cells by introducing a mutation at a specific genomic locus. Murine embryonic stem cells (ES cells) have been isolated that can be genetically manipulated in cell culture and reintroduced into blastocysts, giving rise to chimeric animals. Chimeras may transmit the mutation to their offspring, which will then be heterozygous for the mutation. The mutation can then be bred to homozygosity, enabling the analysis of the mutational phenotype both at the level of the whole animal and at the cellular and molecular level by analyzing cell lines or tissues derived from animals homozygous for the desired mutation. An important task in gene targeting experiments is to identify the rare targeted events over the background of random integrations. Most protocols include the use of a Neo cassette in the targeting vector disrupting an exon of the gene of interest and enabling a positive selection for cells that have stably integrated the transfected DNA into the genome.
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U2 - 10.1016/S0076-6879(94)37076-1
DO - 10.1016/S0076-6879(94)37076-1
M3 - Article
C2 - 7935011
AN - SCOPUS:0028048178
VL - 237
SP - 366
EP - 386
JO - Methods in Enzymology
JF - Methods in Enzymology
SN - 0076-6879
IS - C
ER -