ANNUAL REPORT - Department of Biotechnology

intra seminiferous tubular space of the testis and
electroporated males were mated with the wild type
females. The transgene was successfully
propagated to F3 generation as evidenced by PCR
as well as Southern blotting analysis of genomic
DNA. In another study for the development of cloned
embryos, enucleation technique in mice and buffalo
has been standardized using various somatic cells.
The cumulus cells were found to be best suitable for
production of cloned embryos in both the species.
Transgenic cloned mice were successfully produced
r
using granulosa cells from Neo mice and its
presence in cloned embryos were analyzed by PCR.
Non GFP as well as GFP expressing cloned buffalo
embryos were also produced.
At IISc, Bangalore, EGFP-expression pattern was
evaluated to study transgene expression during
embryo development in mice during the entire period
of oogenesis and spermatogenesis. In the EGFPtransgenic
female, oocytes exhibited green
fluorescence during the entire meiotic maturation
process. EGFP trangene was expressed during fetal
oogenesis and the expression of the transgene
persisted in embryos, which inherited the transgene.
EGFP-expression was detected in 100% oocytes
and embryos.
Buffalo Genomics:
A multicentric programme on Buffalo Genomics was
initiated with an emphasis on identification of genes
of economic importance. Significant achievements
are as follows :
Radiation hybrid mapping panel and DNA markers for
creation of buffalo genome map is being developed at
CCMB, Hyderabad. So far, 81 radiation hybrid clones
of buffaloes with the background of Chinese hamster
genome were created. Besides this, 598
microsatellite markers were developed which would
be used for mapping a radiation panel. An additional,
74 putative hybrid clones were isolated and are being
tested for contents of buffalo genome. To develop
expressed sequence tags(EST) markers for
buffaloes, 284 cattle EST primers pairs on buffalo
genomic DNA were tested. Out of these, unique
fragments from 167 (59%) primers pairs could be
amplified and sequenced. To increase the efficiency
of buffalo EST development, a database of primer
pairs for 1960 potential buffalo ESTs was created.
Out of these, 1023 ESTs have been validated. At
present, more than one thousand DNA markers
(microsatellite and ESTs) are available for creation of
a radiation hybrid map of buffaloes.
Characterization and mapping of fertility related
hormone / hormone receptor genes in Buffalo was
attempted at NDRI, Karnal. The full coding part of the
buffalo FSHβ gene corresponds well to the GenBank
reported sequence of buffalo. The mutation study
revealed a total of eight substitutions of nucleotides in
the FSHβ gene coding part. Out of these, five
pertained to amino acid changes and three were
silent mutations. The FSH receptor gene was
sequenced and conserved motifs and Nglycosylation
sites also corresponded with most of
the other mammalian species. A total of nine
nucleotides substitutions were observed in the
coding part of FSH receptor gene and out of these six
mutations were reported to be silent.
At NBAGR, Karnal, cDNA library from buffalo
mammary gland tissues (lactating & non-lactating)
was screened for maximally expressed genes.
Sequence information for 764 buffalo mammary
gland ESTs from lactating and non-lactating stages
was generated and charaterized. Out of these 222
EST sequences were submitted to GenBank/ dbEST
data-base. Complete sequence characterization of
two important buffalo mammary gland derived genes
viz. Osteopontin and Beta-casein genes was carried
out and compared with other livestock species.
Buffalo specific cDNA probes with respect to
mammary derived genes were generated to screen
the redundancy and expression profile of individual
genes in mammary gland cDNA library.
Genes regulating embryonic survival and maternal
recognition of pregnancy in buffaloes were
characterized at IVRI, Izatnagar. The complete
coding sequence of Uterine Milk Protein (UTMP)
cDNA of 1309 bp was cloned and characterized. The
85 Research and Development