Fogarty Report
- 1. Report of work carried out from May 1st to Oct 24th , 2005 in the Dept of Cell Biology at CRCRH, University of Virginia, Charlottesville, USA, submitted to the Govt. of India, by Dr Manjeet K. Sharma, Assistant Director, Neuroendocrinology Department, NIRRH, Mumbai, India, during the tenure of Fogarty fellowship (NIH). Expanding male contraceptive choices is a major research interest of NIRRH. I have been involved in understanding the regulatory role of gonadotropins in spermatogenesis since 1979, in the department of Neuroendocrinology. The current attempts at NIRRH focus on the identification and characterization of target proteins for male contraception. However, the functions of putative targets will first have to be proved in in vivo genetic models. Drugs can then be designed to inhibit the functional domains of the target protein for contraception. In this context, the Fogaarty International Fellowship offered by NIH, USA, for on-the-job training and development of gene knockout mice, was availed of for one year. A further extension was offered by NIH pending sanction from the home institution, in order to complete the work and for eventual transfer of technology to the home institution. Permission was granted for six months and availed of. Essentially, the project involved: 1. Development of gene targeting construct by cloning 2. Development of probes by cloning 3. Preparation of targeting/knockout vector in bulk 4. Targeted deletion of a specific gene in embryonic stem cells by homologus recombination 5. Screening of transformed embryonic stem cells 6. Development of chimeric mice through blastocyst injections of transformed embryonic stem cells 7. Breeding of germline chimeras to hetero- and homozygosity 8. Phenotypic analysis of chimeric mice Objective 1 was successfully achieved at the end of the year (May 1st 2004 to March 31st 2005).
- 2. Title of Project: Cloning and characterization of sperm targets for male contraceptives. Subtitle: Development of a targeting construct for the development of CABYR knockout mice. Resume: CABYR, a calcium binding, tyrosine-regulated sperm fibrous sheath protein has been implicated in the acquisition of motility during capacitation. However, this role needs to be established unequivocally in an appropriate animal model, since the protein is conserved from mouse to human, in order for it to be used as a target for male contraception. The development of cabyr knockout mice has been undertaken with this objective in mind. During the course of this year, an effort was made to develop a targeting construct, which would be able to knockout the mouse cabyr gene and enable the analysis of the impact of knockout on sperm motility and fertilizing ability. The knockouts are expected to generate a phenotype with defects in sperm motility. Mouse cabyr gene is about 14kb in length, located on chromosome 18. The gene is composed of six exons and five introns. Of the four reported splice variants of this gene, AF359382 encodes the functionally active, calcium binding form A. Therefore, we adopted the strategy to knockout exon 4 (1kb), which encodes about 85% of cabyr A. Deletion of the splice junction between exon 4 and intron 3 was part of the strategy to delete cabyr B which is encoded by the splice variants of the gene. Towards this aim, suitable primers were designed to amplify from SvJ mouse genomic DNA, two 3-4 kb fragments of the gene around the exon 4 and clone these into TOPO vectors. The DNA fragments were confirmed to be sufficiently unique so as not to bear homology to other regions of the mouse genome. These DNA fragments referred to as 3’ arm-3.5 kb and and 5’ arm-3.9 kb were first cloned into TOPO vectors. The plasmids were amplified in E coli. Positive colonies with cloned plasmid DNA were screened. Plasmids were extracted and identified by restriction analysis prior to getting the sequences confirmed. Subsequent to that, the sequence of the entire length of cloned DNA was confirmed by primer walk. The plasmids with 100% correct sequence of cloned DNA were used for subcloning. After cloning the two arms, the 5’ arm was reamplified by PCR from 5’ TOPO clone with another set of primers carrying the restriction sites for Xho 1 and Xma 1 enzymes. The 3’ arm was likewise reamplified from 3’ TOPO clone with another set of primers carrying the restriction sites for Asc1 and Kpn1 enzymes. The two new arms carrying the restriction sites were again subcloned into TOPO vector. The cloned plasmids were amplified in E coli and screened for positive colonies. The subcloning was again confirmed first by restriction analysis. The correct plasmids with subcloned
- 3. DNA were sent for sequence confirmation. Each of the two arms was excised out of the vector with the restriction enzymes used for subcloning. The arms were ligated into the multiple cloning sites of a novel targeting vector pCJUKO developed in the Center, one at a time, after linearizing these with the same restriction enzymes to create cohesive ends. The technique involves extensive screening of colonies at each step to identify the positive colonies carrying the cloned DNA arms. Once identified, the sequences of the whole arms are confirmed by microsequencing. The 5’arm has been ligated into the targeting vector. The ligation of 3’ arm into the targeting vector carrying the 5’ arm has also been confirmed. Glycerol stocks of all the positive clones for both arms in TOPO and pCJUKO vectors have been made and stored at -70°C. Future agenda: After the arms have been cloned, the targeting vector has to be transfected into SvJ mouse embryonic stem cells by electroporation for integration of the targeting vector in one of the two alleles, at the precise locus, by homologus recombination. An effort was also made to get familiar with the ES cell culturing technique at the Center. Attempt was also made to standardize the southern hybridization technique which will be used to identify the ES clones carrying the integrated insert with a 3’ dummy arm probe generated by PCR. Once the positive clones can be identified, these will be introduced into host murine embryos which will be allowed to grow in murine foster mothers. Once the progeny are born, animals will be screened for hemizygous mutants which will then be crossbred to produce homozygous mutants, lacking the exon 4. The genotyping of the mutants will be done in tail biopsies by an initial PCR screen for the targeted inserts followed by southern hybridizations with 3’ and 5’ arm probes. Attempt was made to standardize the PCR for the 3’ and 5’ arms in R mouse tail biopsies. The animals so bred will be used for functional analysis.
- 4. Objectives 2 and 3 were successfully achieved at the end of the 5 month extension period from May 22nd to October 24th , 2005. Title of Project: Cloning and characterization of sperm targets for male contraceptives. Subtitle: Development of a targeting construct for the development of CABYR knockout mice. Resume: During the 6 month extension period, the targeting vector was amplified in bulk in E coli. The knockout vector plasmid was extracted and analysed by restriction analysis to drop out the 5’-3.9 kb and 3’-3.5 kb arms on agarose gel. The vector was linearized with Not1 restriction enzyme overnight. The linearization of an aliquot was checked on agarose. The linear vector was extracted with phenol-chloroform-isoamyl alcohol. The vector was salt precipitated with sodium acetate and chilled absolute alcohol overnight at -70°C. The precipitate was washed with 70% alcohol, solubilised in water/TAE buffer, quantitated, and stored at -20°C. The linear vector will be transfected in embryonic stem cells by electroporation for a heterozygous knockout of cabyr gene. Multiple probes for screening the transfected cells by southern hybridization were prepared by cloning in TOPO vector. Two probes for the detection of 5’arm and one probe for 3’ arm were cloned from 129SvJ murine DNA into TOPO vector. Plasmids were amplified in E coli cells and positive colonies identified. Plasmids were extracted and cloning confirmed by restriction analysis. The sequence of the plasmids with insert was given for confirmation. Glycerol stocks were prepared and stored at -70°C. Probes were cut from amplified plasmids on agarose gels and purified. Purified probes were quantitated on gel and spectrophotometer and stored at -20°C. Screening strategy by PCR was also standardized. Three sets of probes were cloned in TOPO vector using multiple primers for wild type and mutant cabyr gene in embryonic stem cells. The probes were amplified in E coli and plasmids extracted for conformation of cloning and sequence. Two sets of primers were identified from positive clones to detect the wild type and mutant genes in embryonic stem cells by PCR. The primers were stored at -20°C for future use. At least one attempt was made at electroporation of targeting vector in R1S 129SvJ mouse stem cells, grown on irradiated, grown on irradiated mouse stem cells expressing Neomycin resistance. The whole procedure lasted for 6 weeks. However, none of the stem cells survived geneticin challenge. Few clones that survived were too small for picking, freezing and expansion. Therefore, the experiment to extract DNA from surviving clones could not be done. The
- 5. electroporation needs to be repeated several times to get results. The electroporator failed in this particular experiment. The remaining objectives 4-8 needed extensive studies over a period of time for training and results, before the knockout results in a chimera fit for phenotype analysis. This would have required availing further extension offered by NIH.