Staff:

Group leader : RAFFAELE GEREMIA

Universita' degli Studi di ROMA "Tor Vergata"
MEDICINA E CHIRURGIA
DIPARTIMENTO DI SANITA' PUBBLICA E BIOLOGIA CELLULARE

1. PELLEGRINO ROSSI UNIVERSITA' DI ROMA TOR VERGATA DIPARTIMENTO DI SANITA' PUBBLICA E BIOLOGIA CELLULARE
2. DAVIDE LAURO UNIVERSITA' DI ROMA TOR VERGATA MEDICINA INTERNA

Research activity

Once the human gene has been identified and/or characterized, the next step will be to clone the murine homologous in order to generate mutant animals. As stated previously, two approaches which will be followed: the use of the Cre-lox and the knock-in technologies. Both these two techniques are derived from the homologous recombination strategy which allows to modify a genomic locus through the introduction of a manipulated DNA vector sharing at least 6kb of homology with the wild type DNA locus in totipotent stem cells, the embryonic stem cells (ES).
Cre-lox. By using this approach, which has also been referred as conditional knock-out, it is possible to delete any given gene in a particular or in a subset of tissues of the manipulated animal. The first step will require the isolation of the murine gene which is under investigation. Once the genomic sequence is obtained and cloned, two short DNA sequences, named lox sequences, will be placed within the intronic regions of the gene without affecting its structure and functions. The lox sequences are viral sequences which are recognized by the viral enzyme recombinase. Recombinase binds the lox sequences and, keeping them paired, cuts out all the genomic region in between the two loxes. Then, two selection genes will be introduced in the construct (positive selector: neomycin; negative selector thymidine kinase) in order to properly recombine the ES genomic locus. Once the recombined ES clones will be obtained, chimaeric animals will be generated by injecting the ES cells into wild type blastocysts. The chimaeras will be then bred to wild type animals to generate heterozygous and then finally homozygous lox animals. At this point the lox animals will be ready to be inter crossed to transgenic animals carrying the viral recombinase under the control of a mouse tissue specific promoter. The descendant mice will display genomic deletions only in the tissues where the recombinase enzyme will be expressed.
Knock-in. By using this technology it is possible to introduce point mutations in the coding sequence of any particular gene. Also in this case the first step will require the isolation and cloning of the murine genomic locus where the gene to be investigated maps. A point mutation will be then introduced in the coding sequence of the gene in a particular domain which is known to mediate cellular signalling or in domains which need to be investigated. After the mutation is introduced, the construct will be further modified by inserting two selection genes (positive selector: neomycin; negative selector thymidine kinase) and then transfected in the ES cells. Once the selected ES clones will be obtained with the locus correctly recombined, chimaeric mice will be generated. As for the cre-lox chimaeras, the knocked-in chimaeras will be crossed to wild animals to obtain heterozygous mice and then, from these, homozygous animals. The homozygous animals will display a phenotype which will reflect the impairment of the pathway which has been mutated.
Transgenic, "knock-out", "conditional knockout", and "knock-in" animals will be evaluated by both functional and morphological studies. The latter will be performed by analyzing either in autoptic or bioptic specimens the general architecture, the differentiative state, the possible modifications in immunohistochemical characteristics of specific tissues, by using both optical and electron microscopic techniques. Functional studies will involve both analysis of behavioural alterations of the experimental animals, and also in vitro studies on tissue specimens and/or on specific cell populations microdissected from different tissues and/or organs.

Financial support

Amount (ML) 125
Source(s) MURST; CNR; Telethon

Five recent papers

1) ALBANESI C., GEREMIA R., GIORGIO M., DOLCI S., SETTE C., AND ROSSI P. A cell- and developmental stage-specific promoter drives the expression of a truncated c-kit protein during mouse spermatid elongation. Development 122:1291-1302, 1996.
2) SETTE C., BEVILACQUA A., BIANCHINI A., MANGIA F., GEREMIA R., AND ROSSI P. Parthenogenetic activation of mouse eggs by microinjection of a truncated c-kit tyrosine kinase present in spermatozoa. Development 124:2267-2274, 1997.
3) SETTE C., BEVILACQUA A., GEREMIA R., AND ROSSI P. Involvement of phospholipase C g1 in mouse egg activation induced by a truncated form of the c-kit tyrosine kinase present in spermatozoa. J. Cell Biol. 142:1063-1074, 1998.
4) SETTE C., BARCHI M., BIANCHINI A., CONTI M., ROSSI P., AND GEREMIA R. Activation of the mitogen-activated protein kinase ERK1 during meiotic progression of mouse pachytene spermatocytes. J. Biol Chem., 274:33571-33579, 1999.
5) SAMMARCO I., GRIMALDI P., ROSSI P., CAPPA M., MORETTI C., FRAJESE G., AND GEREMIA R. Novel point mutation in the splice donor site of exon-intron junction 6 of the androgen receptor gene in a patient with partial androgen insensitivity sindrome. J. Clin. Endocrinol. Metab., 85:3256-3261, 2000.