21 Transgenie Animals
331
Future perspectives
One of the major problems in transgenic research is the relative limitation in
the size ofthe constructs. The recentlyused cloningvectors limit the size of a
construct to approximately 40 kb. Therefore in some experiments neither
the full genomic sequence nor putative regulatory elements can be included
into the construct. Besides the fact that appropriate regulation and tissue
specificity cannot be obtained with these constructs, the short "buffering
zone" between transgene and endogenous DNA leads to positioning effects
as mentioned above.
A new approach in transgenic research that seems tobe able to solve this
problems is the use of yeast artificial chromosomes (YACs). Recently, a
number of experiments have been reported. Jakobovits et al. introduced
a 670-kb YAC, containing the human hypoxanthine phosphoribosyl transferase gene into ES cells by spheroblast fusion (59) resulting in appropriate
development oftransgenic mice and correct gene regulation. Even integration oflarge amounts of additional yeast DN A was not detrimental to ES cell
growth or development of mice.
Schedl et al. generated a transgenic mouse by direct pronuclear injection
of a construct containing the 80 kb mouse tyrosinase geneplus 155 kb of
flanking sequences. They reported copy-number-dependent and positionindependent expression of the transgene. Furthermore, the transgene
showed the normal tissue-specific pattern (60).
Choi and co-workers reported a transgenic mouse containing an 85 kb
fragment ofthe human heavy chain immunoglobulin gene (61) engineered
by transfection of ES cells followed by blastocyst injection. Serum levels of
human J..l-chains were very low compared with endogenaus mouse IgM,
probably due to competition from the endogenaus heavy chain gene.
The authors suggest that larger "buffering sequences" flanking the gene
would solve this problem.
Peterson et al. (62) reported a mouse, containing an intact 82 kb human
ß-globin locus flanked by a 5' sequence of 148 kb. The construct was inserted into the mouse genome using direct microinjection. Since large molecules such as YACs aresensitive to mechanical shearing, they used a solution of high ionic strength (100 mM NaCl, according to Gnirke et al. (63),
which served as a protective agent. They reported correct developmental
regulation of the gene-locus and expression in the appropriate tissue-specific pattern.
Another approach to obtain tissue-specificity in transgenes is to link the
structural gene to a promoter of known tissue-specificity. One example is
the specific expression in heart-muscle of genesthat are linked to the reg-
331
Future perspectives
One of the major problems in transgenic research is the relative limitation in
the size ofthe constructs. The recentlyused cloningvectors limit the size of a
construct to approximately 40 kb. Therefore in some experiments neither
the full genomic sequence nor putative regulatory elements can be included
into the construct. Besides the fact that appropriate regulation and tissue
specificity cannot be obtained with these constructs, the short "buffering
zone" between transgene and endogenous DNA leads to positioning effects
as mentioned above.
A new approach in transgenic research that seems tobe able to solve this
problems is the use of yeast artificial chromosomes (YACs). Recently, a
number of experiments have been reported. Jakobovits et al. introduced
a 670-kb YAC, containing the human hypoxanthine phosphoribosyl transferase gene into ES cells by spheroblast fusion (59) resulting in appropriate
development oftransgenic mice and correct gene regulation. Even integration oflarge amounts of additional yeast DN A was not detrimental to ES cell
growth or development of mice.
Schedl et al. generated a transgenic mouse by direct pronuclear injection
of a construct containing the 80 kb mouse tyrosinase geneplus 155 kb of
flanking sequences. They reported copy-number-dependent and positionindependent expression of the transgene. Furthermore, the transgene
showed the normal tissue-specific pattern (60).
Choi and co-workers reported a transgenic mouse containing an 85 kb
fragment ofthe human heavy chain immunoglobulin gene (61) engineered
by transfection of ES cells followed by blastocyst injection. Serum levels of
human J..l-chains were very low compared with endogenaus mouse IgM,
probably due to competition from the endogenaus heavy chain gene.
The authors suggest that larger "buffering sequences" flanking the gene
would solve this problem.
Peterson et al. (62) reported a mouse, containing an intact 82 kb human
ß-globin locus flanked by a 5' sequence of 148 kb. The construct was inserted into the mouse genome using direct microinjection. Since large molecules such as YACs aresensitive to mechanical shearing, they used a solution of high ionic strength (100 mM NaCl, according to Gnirke et al. (63),
which served as a protective agent. They reported correct developmental
regulation of the gene-locus and expression in the appropriate tissue-specific pattern.
Another approach to obtain tissue-specificity in transgenes is to link the
structural gene to a promoter of known tissue-specificity. One example is
the specific expression in heart-muscle of genesthat are linked to the reg-