Gene targeting in the mouse nervous system

pre-printOur understanding of the development, connectivity and function of the nervous system has been facilitated by gene targeting technology. Here we summarize the historic background and the current state of this experimental approach with specific regard to neuroscience research. I. The Pioneering Experiments. Random mutagenesis is a very powerful method for elucidating gene function in simpler model organisms. Given the size of the genome and slow reproductive cycles, however, a more direct approach is required for mammalian models. This need was met in the early 1980's by establishing gene targeting in embryonic stem cells, later nicknamed mouse knockout. This technology was born at the confluence of two lines of experimentation: Firstly, the capacity of most mammalian somatic cells to carry out homologous recombination between endogenous loci and exogenous DNA was discovered. Secondly, pluripotent embryonic stem cell lines have been established that maintained the ability to intermingle with the early mouse embryo and contribute to the germline, thereby conferring heritability, even after extensive culturing in vitro

Establishment of mammalian cell lines containing multiple nonsense mutations and functional suppressor tRNA genes.

Journal ArticleWe describe the generation of mammalian cell lines carrying amber suppressor genes. Nonsense mutants in the herpes simplex virus thymidine kinase (HSV tk) gene, the Escherichia coli xanthine-guanine phosphoribosyl transferase (Eco-gpt) gene and the aminoglycoside 3' phosphotransferase gene of the Tn5 transposon (NPT-II) were isolated and characterized. Each gene was engineered with the appropriate control signals to allow expression in both E. coli and mammalian cells. Expression in E. coli made possible the use of well developed bacterial and phage genetic manipulations to isolate and characterize the nonsense mutants. Once characterized, the nonsense mutants were transferred into mammalian cells by microinjection and used, in turn, to select for amber suppressor genes. Xenopus laevis amber suppressor genes, prepared by site-specific mutagenesis of a normal X. laevis tRNA gene, were microinjected into the above cell lines and selected for the expression of one or more of the amber mutant gene products. The resulting cell lines, containing functional amber suppressor genes, are stable and exhibit normal growth rates

Duplication of the Hoxd11 gene causes alterations in the axial and appendicular skeleton of the mouse.

Journal ArticleThe Hox genes encode a group of transcription factors essential for proper development of the mouse. Targeted mutation of the Hoxd11 gene causes reduced male fertility, vertebral transformation, carpal bone fusions, and reductions in digit length. A duplication of the Hoxd11 gene was created with the expectation that the consequences of restricted overexpression in the appropriate cells would provide further insight into the function of the Hoxd11 gene product. Genetic assays demonstrated that two tandem copies of Hoxd11 were functionally indistinguishable from the normal two copies of the gene on separate chromosomes with respect to formation of the axial and appendicular skeleton. Extra copies of Hoxd11 caused an increase in the lengths of some bones of the forelimb autopod and a decrease in the number of lumbar vertebrae. Further, analysis of the Hoxd11 duplication demonstrated that the Hoxd11 protein can perform some functions supplied by its paralogue Hoxa11. For example, the defects in forelimb bones are corrected when extra copies of Hoxd11 are present in the Hoxa11 homozygous mutant background. Thus, it appears that Hoxd11 can quantitatively compensate for the absence of Hoxa11 protein, and therefore Hoxa11 and Hoxd11 are functionally equivalent in the zeugopod. However, extra copies of Hoxd11 did not improve male or female fertility in Hoxa11 mutants. Interestingly, the insertion of an additional Hoxd11 locus into the HoxD complex does not appear to affect the expression patterns of the neighboring Hoxd10, -d12, or -d13 genes

Mice lacking endothelial ACE: normal blood pressure with elevated angiotensin II

Journal ArticleRecently, the concept of local renin-angiotensin systems (RAS) capable of generating angiotensin II apart from the circulation has received considerable attention. To investigate this, we generated ACE 1/3 mice in which one allele of ACE is null and the second allele was engineered to express ACE on the surface of hepatocytes. ACE 1/3 mice express no endothelial ACE and lack ACE within the lungs. Their kidneys contain <7.8% the enzyme levels present in control mice. Plasma conversion of angiotensin I to angiotensin II was 43.3% normal. The baseline blood pressure and renal function of the ACE 1/3 mice were normal, probably as a function of a marked increase of both plasma angiotensin I and angiotensin II. When exposed to 2 weeks of a salt-free diet (a stress diet stimulating the RAS), blood pressure in ACE 1/3 mice decreased to 92.3+2.0 mm Hg, a level significantly lower than that of wild-type control mice. The ACE 1/3 mice demonstrate the plasticity of the RAS and show that significant compensation is required to maintain normal, basal blood pressure in a mouse with an impaired local vascular and renal RAS. (Hypertension. 2003;41:313- 321.

Isolation and characterization of Caenorhabditis elegans DNA sequences homologous to the v-abl oncogene.

Journal ArticleDNA sequences homologous to the v-abl oncogene were isolated from a Caenorhabditis elegans genomic library by their ability to hybridize with a v-src probe. The DNA sequence of 2465 nucleotides of one clone was determined. This region corresponds to the 5' protein kinase domain of v-abl plus approximately equal to 375 base pairs toward the 3' end. Four potential introns were identified. The homology between the deduced amino acid sequence of the C. elegans clone and that of the 1.2-kilobase-pair protein kinase region of v-abl is 62%. The tyrosine residue corresponding to the tyrosine that is phosphorylated in the v-src protein is conserved in the C. elegans sequence. When 95 amino acids around this tyrosine were compared with the corresponding sequences of Drosophila c-abl, v-abl, and v-src, the identities were 83%, 79%, and 56%, respectively. Hybridization of the cloned DNA with C. elegans poly(A)+ RNA revealed a major transcript of 4.4 kilobases

Hoxb8 is required for normal grooming behavior in mice.

Journal ArticleRepertoires of grooming behaviors critical to survival are exhibited by most animal species, including humans. Genes that influence this complex behavior are unknown. We report that mice with disruptions of Hoxb8 show, with 100% penetrance, excessive grooming leading to hair removal and lesions. Additionally, these mice excessively groom normal cagemates. We have been unable to detect any skin or PNS abnormalities in Hoxb8 mutants. These observations suggest that the excessive, pathological grooming exhibited by these mice results from CNS abnormalities. Consistent with this interpretation, we demonstrate Hoxb8 expression in regions of the adult mouse CNS previously implicated in the control of grooming. The aberrant behavior observed in Hoxb8 mutants is not unlike that of humans suffering from the OC-spectrum disorder, trichotillomania. Interestingly, Hoxb8 is expressed in regions of the CNS known as the "OCD-circuit

Hox11 paralogous genes are essential for metanephric kidney induction

Journal ArticleThe mammalian Hox complex is divided into four linkage groups containing 13 sets of paralogous genes. These paralogous genes have retained functional redundancy during evolution. For this reason, loss of only one or two Hox genes within a paralogous group often results in incompletely penetrant phenotypes which are difficult to interpret by molecular analysis. For example, mice individually mutant for Hoxa11 or Hoxd11 show no discernible kidney abnormalities. Hoxa11/Hoxd11 double mutants, however, demonstrate hypoplasia of the kidneys. As described in this study, removal of the last Hox11 paralogous member, Hoxc11, results in the complete loss of metanephric kidney induction. In these triple mutants, the metanephric blastema condenses, and expression of early patterning genes, Pax2 and Wt1, is unperturbed. Eya1 expression is also intact. Six2 expression, however, is absent, as is expression of the inducing growth factor, Gdnf. In the absence of Gdnf, ureteric bud formation is not initiated. Molecular analysis of this phenotype demonstrates that Hox11 control of early metanephric induction is accomplished by the interaction of Hox11 genes with the pax-eya-six regulatory cascade, a pathway that may be used by Hox genes more generally for the induction of multiple structures along the anteroposterior axis

Choose your target.

Journal ArticleThe technology of modifying endogenous genes has recently been extended from mice to Drosophila and sheep. Concurrently, genomic sequencing is uncovering thousands of previously uncharacterized genes. Armed with today's technologies, what are our best options for delineating the functions of these new genes