Isolation of cosmid clones with testase 2 genomic DNA

The mouse RZPD (The Resource Center and Primary Database) genomic library 129 ola was screened using a fragment of testase 2 cDNA (Tes2) and 2 positive clones were obtained:

MPMGc121H24624Q2 and MPMGc121H20723Q2. These two cosmid clones were digested with restriction enzymes, blotted to Hybond C membrane and hybridized with 5’probe (Figure 3.48 A). Cosmids showed slightly different restriction patterns indicating different genomic content of both clones. Clone MPMGc121H20723Q2 was used for generation of knock out construct. During the time knock out construct was generated it was not known that there are two copies of the testase 2 gene. Sequencing results and restriction analysis have suggested that cosmid MPMGc121H20723Q2 contains testase 2α genomic fragment whereas clone MPMGc121H24624Q2 contains genomic fragment with testase 2β.

Figure 3.48 (A) Radioactive hybridization of cosmid clone H24624Q2 and H20723Q2 digested with different restriction enzymes. (B) Restriction digestion map of testase 2 genomic DNA and fragments which were cloned, abbreviations are: B: Bam HI; E: Eco RI; H: Hind III; P: Pst I; SI: Sst I; X: Xba I;

Xh: Xho I.

B SI P P X SI P B X

Exon 1 Exon 2

Fragment P Fragment X

X E Xh/X P P/X S S/X H H/X X E Xh/X P P/X S S/X H H/X

H24 H20

A

B

A knock out construct was generated in which 5’wing 2 kb Pst I fragment was cloned into Sal I/Cla I site of pTKNeo vector and 3’wing 5 kb Xba I fragment was cloned into Spe I/ Bam HI site of the targeting vector (Figure 3.49). After homologous recombination genomic fragment of about 1.8 kb was replaced by Neomycin resistance gene. Neomycin was used as positive selection marker and two copies of herpes virus Thymidine kinase (Tk) as a negative selection marker. The correct orientation of both 5’ and 3’ wings was confirmed by sequencing of the construct with vector specific primers pTKNf, pTKNr, pTKr.

Figure 3.49 Schematic representation of the targeting strategy. Wildtype testase 2 locus (A), targeting vector (B) and mutated allele (C). Parts of exon 1 and 2 were replaced by the Neomycin resistance gene cassette. External probe is marked as green bar, which enables to recognize an 12.3 kb Bam HI fragment in wildtype DNA and a 3.1 kb fragment in recombinant DNA. The gray boxes represent exons of testase 2 gene, green boxes stand for Thymidine kinase and blue box stands for Neomycin. KOT2fp, KOT2rp and NeoRI are the primers used for genotyping. The restriction site abbreviations are: B: Bam HI; C: Cla I; P:

Pst I; SI: Sst I; SII: Sst II; Sp: Spe I; S: Sal I; X: Xba I; N: Not I; Ext: external probe.

B SI P P

Neo

Sp X SI P B X N SII X SI P B X

S SI P P C B

B S SI P P C B

Tk Tk

Neo

SpX SI P B X Exon 1 Exon 2

Exon 1

Wild type allel 12.3 kb

Mutant allel 3.1

NeoRI Ext KOT2fp KOT2rp

3.2.3.2 Generation of the 5’external probe

A fragment about 0.5 kb was amplified by PCR using expT2fp and expT2rp primers.

Fragment is localized upstream of testase 2 gene and was designed to detect Bam HI wildtype fragment in Southern blot hybridization (Figure 3.49). This PCR product was subcloned into pGEM-TEasy and cut out with Eco RI. The 0.5 kb fragment was extracted from the agarose gel and used as 5’ external probe for hybridization in the Southern blot with DNA extracted from the recombinant ES-clones. Generated external probe was detecting exclusively genomic fragment belonging to testase 2 α.

3.2.3.3 Electroporation of the RI ES-cells and screening of ES-clones for homologous recombination events

The testase 2 targeting vector was linearised by digestion with Sst II restriction enzyme and 50 µg of linearised DNA was electroporated into RI embryonic stem cell line as described in section 2.2.20.1. The cells were plated on fibroblast feeder layer and after 10 days of selection individual Neomycin resistant clones were picked in 24 well plates and replicated. Genomic DNA was prepared from these ES clones for Southern blot analysis. DNA of individual ES clones was digested with Bam HI and electrophoresed into 1% agarose gels and blotted onto Hybond C membranes. The blots were hybridised with ³²P-labelled 0.5 kb 5’ external probe.

In the case of homologous recombination event two bands were expected, a wildtype allele of 12.3 kb and a recombinant allele of 3.1 kb. However in the event of a random integration, only wildtype allele will be detected (Figure 3.50). By screening of more than 200 clones no homologues recombination was found. Further transfections were not performed due to the fact, that there is gene duplication and knocking out both copies, would be impossible because of the close distance between genes. Furthermore, knocking out only one copy would not give clear information about gene function. Other question also arises: if these two genes are functional or these are pseudogenes.

Figure 3.50 Genomic Southern blot analysis. Genomic DNA of ES clones was digested with Bam HI, separated on 1 % agarose gel and transferred onto Hybond C membrane. The blot was hybridized with radioactive labeled 0.5 kb external probe. In case of wildtype allele (ES clones 1,2 and 3) one band of 12.3 kb was observed and in case of recombinant allele two bands would be observed, in addition to wildtype allele, a recombinant allele of 3.1 kb.

Wild type allel (12.3 kb)

4 DISCUSSION

ADAM it is an interesting family of proteins, characterized by having two potential activities, proteolytic and adhesive. Many of the ADAM family genes are expressed in the testis indicating the important role of these molecules in germ cell differentiation and in participation in gamete interaction during fertilization. The functions of two murine proteins expressed in testis, namely cyritestin and fertilin β were already elucidated, indicating their role in the fertilization process (Shamsadin et al., 1999; Cho et al., 1998). It requires further investigations to understand the functions of all testis specifically expressed ADAMs in spermatogenesis and fertilization. We focused our work on two germ cell specific genes, ADAM 27 and testase 2, and their possible functions. In the first part of this thesis, the results concerning ADAM 27 gene are discussed. Its genomic structure, chromosomal localization and expression pattern are characterized. Two different types of approaches in vitro (sperm-egg binding assays and yeast two hybrid screen) and in vivo (knock out and transgenic animal models) are described with their implications for ADAM 27 function. In the second part of the thesis I discuss duplication, genomic structure and expression of testase 2 α and testase 2 β genes. And finally, the generation of the knock out construct, difficulties in producing testase 2 α and β null mice, and possible functions of testase 2 α and β proteins are discussed.