the presence of the DHFR gene has not 
been confirmed with DNA hybridization 
studies and. until such experiments are 
reported, the efficiency of the calcium 
phosphate procedure is uncertain. 
Shortcomings of chemical techniques. 
If a chemical technique for gene transfer 
were used in a protocol designed for 
humans, the predicted results appear dis- 
couraging. Recovery from hone marrow 
of approximately 10'“ nucleated cells (of 
which I0 7 to 10* arc stem cellsi can 
routinely be obtained from patients for 
marrow transplantation. F.fficiency of I 
in 10* would mean that only 10 to 100 
stem cells would be transfected. Reinser- 
tion of these cells into the total stem cell 
pool of 10* to It)'* cells would be very 
unlikely to have any noticeable effect on 
a patient's course unless there was an 
extraordinary selective advantage for the 
treated cells. Any human gene therapy 
protocol that uses chemical means for 
transfection would have to establish, 
therefore, that either a few transfected 
stem cells might have a detectable bene- 
ficial effect on the patient's course or 
that the investigator has improved sub- 
stantially the efficiency of the procedure 
for human bone marrow cells. 
Physical Techniques 
Microinjection (20) and electropora- 
tion Ul I are the two principal classes of 
physical techniques. Electroporation. a 
relatively new technique, is the transport 
of DNA directly across a cell membrane 
by means of an electric current. It has 
been used to transfer a variety of genes 
into a number of different cells including 
the immunoglobulin k gene into B cells 
(2/). Its potential for human gene thera- 
py is uncertain. 
Microinjection has been used for a 
number of years and has the advantage 
of high efficiency (up to one cell in five 
injected can be permanently transfect- 
ed!. However, the distinct disadvantage 
is that only one cell at a time can be 
injected. Transfection of a large number 
of hematopoietic stem cells is not feasi- 
ble. Even if a stem cell could be recog- 
nized it would have to be fixed to a slide 
for injecting. The effect of attaching, 
injecting, and subsequent detaching is 
unknown. Microinjection of mouse 
crythrolcukcmia (MEL) cells is difficult, 
although possible (22). and these cells 
arc much easier to manipulate in culture 
than are bone marrow cells. 
Transfer of genes into mouse eggs. An 
area where microinjection has had spec- 
tacular success is in transferring genes 
into fertilized mouse eggs (2.1). Gordon 
XK.TOIII K IVK4 
el ol. (Ml first demonstrated that if plas- 
mid DNA is microinjccted into one of the 
two pronuclci of a recently fertilized 
mouse egg. and the ovum is then placed 
into the oviduct of a pseudopregnant 
female, the egg could develop into a 
normal mouse carrying the plasmid DNA 
in every cell of its body. Furthermore, 
the injected DNA can he transmitted to 
offspring in a normal Mendelian manner. 
Mice carrying an exogenous gene in their 
genome are called "transgenic." 
Hammer el al. (2) used this technique 
to partially correct a mouse with a defect 
in its growth hormone production. By 
attaching a rat growth hormone gene to 
an active regulatory sequence (specifi- 
cally. the promoter that normally directs 
the synthesis of mctallolhioncin mes- 
senger RNA in mice), they obtained a 
recombinant DNA construct that active- 
ly produces growth hormone in the ge- 
netically defective mouse. Although the 
level of growth hormone production is 
inappropriately controlled — that is. in- 
fluenced by signals that normally regu- 
late mctallolhioncin synthesis — these ex- 
periments do show that microinjection 
can be used as a delivery system that can 
put a gene into every cell of an animal's 
body. 
Nonapplicuhility for human*. Should 
the technique of microinjecting a fertil- 
ized egg be employed for human gene 
therapy at the present lime? The answer 
is no on three grounds: the procedure 
has a high failure rate, can produce a 
deleterious result, and would have limit- 
ed usefulness. Microinjcction has a high 
failure rate because the majority of eggs 
are damaged by the microinjection and 
transfer procedures so that they do not 
develop into live offspring. In one recent 
experiment involving microinjcction of 
an immunoglobulin gene (22). 3<X) eggs 
were injected. 192 <M percent) were 
judged sufficiently healthy to be trans- 
ferred to surrogate mothers, only 1 1 (3.7 
percent) proceeded to live birth and f> (2 
percent) carried the gene. These results 
are from a highly experienced laboratory 
in which thousands of identical eggs 
from the same hybrid cross of inbred 
mice have been injected over a number 
of years. The mice were chosen precise- 
ly because they gave the best results for 
gene transfer by microinjcction. Experi- 
ence with attempts to microinjcci growth 
hormone genes into livestock eggs have 
met with a number of major biological 
and technical problems (26). Successful 
gene transfer by microinjcction of human 
eggs, without a long period of trial and 
error experimentation, is extremely un- 
likely. 
Second, microinjcction of eggs can 
produce deleterious results because 
there is no control over where the inject- 
ed DNA will integrate in the genome, 
l.acy el ol. (27) showed that the integra- 
tion of an exogenous rabbit [J-globin 
gene in transgenic mice could sometimes 
occur into a chromosomal location that 
results in expression of the (1-globin gene 
in inappropriate tissue, namely, muscle 
or testes. There have been a number of 
cases reported where integration of mi- 
croinjccted DNA has resulted in a patho- 
logical condition (2/0. Although there is 
no control over where exogenous DNA 
will integrate in any gene transfer proce- 
dure. the damaging effect caused by a 
harmful insertion site could be great 
when it occurs in the egg but may be 
negligible when it occurs in one or a few 
of a large number of bone marrow cells. 
I hird is limited usefulness. Not only is 
it of questionable ethics to experiment 
on human eggs because of the expected 
losses, but even if "success" were ob- 
tained. it would be applicable primarily 
when both parents are homozygous for 
the defect. When the parents are both 
carriers, only one fertilized egg out of 
four would result in an affected child 
(2V(. Since a homozygous defect cannot 
yet be recognized in an ovum, and since 
the procedure itself carries such a high 
nsk. it would be improper to attempt any 
manipulation in this situation. Further- 
more. most of the very serious genetic 
disorders result in infertility (or death 
before reproductive age) in homozygous 
patients. Consequently, there would be 
little use for the procedure even if it were 
available. A different approach for hu- 
man gene therapy is required. 
Expression 
The second criterion for evaluating a 
human gene therapy protocol is that 
there be appropriate expression of the 
new gene in the target cells. Even when a 
delivery system can transport an exoge- 
nous gene into the DNA of the correct 
cells of an organism, it has been a major 
problem to get the integrated DNA to 
function. A vast array of cloned genes 
have been introduced into a wide range 
of cells by the several gene transfer 
techniques discussed above. "Normal" 
expression of exogenous genes is the 
exception rather than the rule. 
Active exogenous promoters in trans- 
genic mice. Microinjcction of fertilized 
eggs with exogenous DNA to obtain 
transgenic mice carrying an expressing 
gene has resulted in several spectacular 
successes, but also in a considerable 
number of unpublished failures. Thus far 
4II< 
Recombinant DNA Research, Volume 12 
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