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.