High efficiency transformation by direct microinjection of DNA into cultured mammalian cells.

Journal ArticleDirect microinjection of DNA by glass micropipettes was used to introduce the Herpes simplex virus thymidine kinase gene into cultured mammalian cells. When DNA was delivered directly into the nuclei of LMTK-, a mouse cell line deficient in thymidine kinase activity, 50--100% of the cells expressed TK enzymatic activity. In contrast, no TK activity could be detected when the DNA was injected into the cytoplasm. The number of injected LMTK- cells capable of indefinite growth in a TK+ selective medium (that is, transformants) depended on the nature of the plasmid DNA into which the HSV-TK gene was inserted. One cell in 500-1000 cells which received nuclear injections with pBR322/TK DNA gave rise to a viable colony when grown in HAT medium (that is, a TK+ selective medium). The transformation frequency increased to one in five injected cells when specific SV40 DNA sequences were also introduced into the HSV-TK plasmid. With the microinjection procedure transformation frequency was relatively insensitive to DNA concentration and did not depend on co-injecting with a carrier DNA. Most of the transformants were stable in nonselective medium as soon as they could be tested

Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells

Journal ArticleWe mutated, by gene targeting, the endogenous hypoxanthine phosphorlbosyl transferase (HPFlT) gene in mouse embryo-derived stem (ES) cells. A specialized construct of the neomycin resistance (NO') gene was introduced into an exon of a cloned fragment of the Hprf gene and used to transfect ES cells. Among the G418' colonies, l/l000 were also resistant to the base analog &thioguanine (&TG). The G418', 8-TGr cells were all shown to be Hprt- as the result of homologous recombination with the exogenous, neo'-containing, Hprf sequences

Introduction: the molecular genetic analysis of mouse development

Journal ArticleThis paper is an introduction of seven different papers presented in "Seminars in developmental biology" on Molecular Genetic Analysis of Mouse Development . The first paper, by Janet Rossant, describes very early mouse development. The second paper, by Frank Conlon and Rosa Beddington provide an intriguing and insightful comparison of gastrulation in Xenopus and mouse. In the third paper, Brigid Hogan reviews the roles of members of the TGF-β super family in mouse development. In the fourth paper, McMahon and his colleagues provide us with a clear overview of the role of the Wnt family members in mouse development. The fifth paper by Mark, Rijli and Chambon, expertly summarized The role of Hox genes in patterning the branchial region of the head. The sixth paper, by St-Onge, Tuello and Gruss, reviews the role of Pax genes in mouse development. The last paper by Elizabeth Robertson in a fascinating report that provides a description of how two growth factors, IGFI and IGFII and their receptor IGF1R interact to control the growth of the embryo and extraembryonic tissues

Hox genes and mammalian development

presentationWe have examined the interactions of Hox genes in forming a cervical vertebrae, hindbrain, and limbs. In each case, it is apparent that individual Hox genes are performing individual functions but that more profound roles are apparent when they act in combination with others Hox genes. The observed interactions suggest that multiple Hox genes function in concert to regulate overlapping sets of target genes. This suggesting is particularly strong in the interactions observed among the group-3 analogous genes in formation of the cervical vertebrae and among Hoxd11, Hoxd12, and Hoxd13 in formation of the autopod. In each case, the cumulative effect of combining multiple mutations is the deletions of structure, resulting from either lack of specification or lack of specification or lack of proliferation of the precursor cells needed to from the structures. Similarly, the combination of HoxaI and HoxbI mutations results in more extensive deletions of anterior structures than is apparent in mice homozygous for either individual mutation. All the results, both of single and combined mutations, are compatible with a role of Hox genes in the early regionalization of the embryo. In the absence of Hox gene functions, formation of the axes and germ cell layers of the embryo still occurs. At this point, the Hox genes are activated to initiate the formation of the embryo by conferring positional value along the major axes of the embryo. Perhaps the most primitive function of Hox genes is the innate ability, through their chromosomal organization, to covet a series of temporal signals into morphological direction, a conversion of time's arrow into a spatial vector

Gene targeting: an historical perspective

Journal ArticleOur entry into what was going to become the field of gene targeting began in 1977. I was experimenting with the use of extremely small glass needles to inject DNA directly into the nuclei of living cells. The needles were controlled by hydraulically driven micro-manipulators and were directed into nuclei with the aid of a microscope. Using this experimental paradigm, I asked myself whether I could introduce a functional gene into cells by injecting the DNA directly into their nuclei. This procedure turned out to be extremely efficient. One in three cells received the DNA in functional form and went on to divide and stably pass the gene onto its daughter cells (Capecchi, 1980). The high efficiency of micro-injection meant that it was now practical to use this technology to generate transgenic mice by the injection of DNA into one-cell zygotes. The embryos were then allowed to come to term by surgical transfer of the zygote to foster mothers. Indeed, this has become a cottage industry in many laboratories throughout the world (Gordon et al, 1980; Costantini and Lacy, 1981; Brinster et al, 1981; Wagner, E.F. et al, 1981; Wagner, T.C. et al, 1981) . However, generation of transgenic animals in this way involves the introduction of exogenous DNA segments at unpredictable locations in the recipient genome, and not targeted genetic alterations at defined sites

Personal view of gene targeting

Book ChapterGene targeting provides the means for creating strains of mice with mutations in virtually any gene.1 First, the desired mutation is introduced into a cloned copy of the chosen gene by standard recombinant DNA technology. The mutation is then transferred to the genome of a pluripotent mouse embryo-derived stem (ES) cell by means of homologous recombination between the exogenous, mutated DNA sequence and the cognate DNA sequence in the ES cell chromosome. By microinjection of ES cells containing the transferred mutation into blastocysts and by allowing the embryos to come to term in foster mothers, we can generate chimeric mice capable of transmitting the mutation to their offspring (germline chimeras). Finally, interbreeding of heterozygous siblings yields animals homozygous for the desired mutation. Figure 1 outlines the steps, from cultured ES cells to chimeric mouse, used to generate mice with targeted mutations

Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes.

Journal ArticleGene targeting--homologous recombination of DNA sequences residing in the chromosome with newly introduced DNA sequences--in mouse embryo-derived stem cells promises to provide a means to generate mice of any desired genotype. We describe a positive nd negative selection procedure that enriches 2,000-fold for those cells that contain a targeted mutation. The procedure was applied to the isolation of hprt- and int-2- mutants, but it should be applicable to any gene

Selective degradation of abnormal proteins in mammalian tissue culture cells.

Journal ArticleThe degradation rates of several missense mutants of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) in mouse L cells are compared to those of the wild-type enzyme. Although the rates of total protein breakdown in the mutant cell lines are identical to that of the parental L cell line, defective molecules of hypoxanthine-guanine phosphoribosyltransferase present in the mutant cell lines are degraded much faster than the wild-type enzyme. The level of defective phosphoribosyltransferase molecules present in the mutant cell lines is inversely proportional to the breakdown rate. This observation indicates that the major factor determining the concentrations of the defective phosphoribosyltransferases is their specific degradation rate. These results strongly support the hypothesis that abnormal proteins are selectively degraded in mammalian cells

Hox group 3 paralogous genes act synergistically in the formation of somitic and neural crest-derived structures.

Journal ArticleHox genes encode transcription factors that are used to regionalize the mammalian embryo. Analysis of mice carrying targeted mutations in individual and multiple Hox genes is beginning to reveal a complex network of interactions among these closely related genes which is responsible for directing the formation of spatially restricted tissues and structures. In this report we present an analysis of the genetic interactions between all members of the third paralogous group, Hoxa3, Hoxb3, and Hoxd3. Previous analysis has shown that although mice homozygous for loss-of-function mutations in either Hoxa3 or Hoxd3 have no defects in common, mice mutant for both genes demonstrate that these two genes strongly interact in a dosage-dependent manner. To complete the analysis of this paralogous gene family, mice with a targeted disruption of the Hoxb3 gene were generated. Homozygous mutants have minor defects at low penetrance in the formation of both the cervical vertebrae and the IXth cranial nerve. Analysis and comparison of all double-mutant combinations demonstrate that all three members of this paralogous group interact synergistically to affect the development of both neuronal and mesenchymal neural crest-derived structures, as well as somitic mesoderm-derived structures. Surprisingly, with respect to the formation of the cervical vertebrae, mice doubly mutant for Hoxa3 and Hoxd3 or Hoxb3 and Hoxd3 show an indistinguishable defect, loss of the entire atlas. This suggests that the identity of the specific Hox genes that are functional in a given region may not be as critical as the total number of Hox genes operating in that region

Hoxc13 mutant mice lack external hair

Journal ArticleHox genes are usually expressed temporally and spatially in a colinear manner with respect to their positions in the Hox complex. Consistent with the expected pattern for a paralogous group 13 member, early embryonic Hoxc13 expression is found in the nails and tail. Hoxc13 is also expressed in vibrissae, in the filiform papillae of the tongue, and in hair follicles throughout the body; a pattern that apparently violates spatial colinearity. Mice carrying mutant alleles of Hoxc13 have been generated by gene targeting. Homozygotes have defects in every region in which gene expression is seen. The most striking defect is brittle hair resulting in alopecia (hairless mice). One explanation for this novel role is that Hoxc13 has been recruited for a function common to hair, nail, and filiform papilla development